The sequences and activities of RegB endoribonucleases of T4-related b
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《核酸研究医学期刊》
Department of Gene Engineering, Institute of Biochemistry, Mokslininku 12, 08662 Vilnius, Lithuania
* To whom correspondence should be addressed. Tel: +370 5 2729146; Fax: +370 5 2729196; Email: rimasn@bchi.lt
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
+AJ439451, AJ490327, AJ496183, AJ488513–AJ488527, AJ606889–AJ606905
DDBJ/EMBL/GenBank accession nos+
ABSTRACT
The RegB endoribonuclease encoded by bacteriophage T4 is a unique sequence-specific nuclease that cleaves in the middle of GGAG or, in a few cases, GGAU tetranucleotides, preferentially those found in the Shine–Dalgarno regions of early phage mRNAs. In this study, we examined the primary structures and functional properties of RegB ribonucleases encoded by T4-related bacteriophages. We show that all but one of 36 phages tested harbor the regB gene homologues and the similar signals for transcriptional and post-transcriptional autogenous regulation of regB expression. Phage RB49 in addition to gpRegB utilizes Escherichia coli endoribonuclease E for the degradation of its transcripts for gene regB. The deduced primary structure of RegB proteins of 32 phages studied is almost identical to that of T4, while the sequences of RegB encoded by phages RB69, TuIa and RB49 show substantial divergence from their T4 counterpart. Functional studies using plasmid–phage systems indicate that RegB nucleases of phages T4, RB69, TuIa and RB49 exhibit different activity towards GGAG and GGAU motifs in the specific locations. We expect that the availability of the different phylogenetic variants of RegB may help to localize the amino acid determinants that contribute to the specificity and cleavage efficiency of this processing enzyme.
INTRODUCTION
RNases play a central role in every aspect of T4 RNA metabolism, including decay of mRNA, conversion of RNA precursors to their mature forms and end-turnover of certain RNAs. Both host and phage endo- and exoribonucleases are involved in processing of phage transcripts (1–3). A host cell contains a large number of distinct RNases approaching as many as 20 members, often with overlapping specificities (4,5). Eight distinct 3' to 5' exoribonucleases, polynucleotide phosphorylase (PNPase), RNase II, RNase D, RNase BN, RNase T, RNase PH, RNase R and oligoribonuclease (5,6), and at least five endoribonucleases, RNases III, E, G, P and I/M (6–8) have been characterized in Escherichia coli. Among these ribonucleases, RNase E has the most prominent effect on the functional and chemical decay of many T4 mRNAs, and therefore it is considered the primary endoribonuclease for phage mRNAs (9). The endoribonuclease E acts throughout all phage development, but in addition the T4 encodes its own endoribonuclease RegB, which participates in the turnover of early mRNAs of T4 (10–14).
The RegB endoribonuclease encoded by phage T4 is a sequence-specific endoribonuclease, which cleaves mRNAs in the middle of sequence GGAG or, in a few cases, in the sequence GGAU (10,11,13). The most efficient cleavages occur in the motifs located in the intergenic regions of early T4 mRNAs. Out of 25 processing sites identified (13,15), 12 occur in Shine–Dalgarno (SD) sequences in such a way making ribosome-binding site non-functional. Primarily recognized examples were observed in the SD regions of the early genes motA, frd and comC (10–12). This suggested the idea that RegB has a general role in the breakdown of T4 early mRNAs. Indeed, in the absence of RegB the chemical half-life of early transcripts of T4 is increased by 4-fold, though their functional half-life is increased slightly by <2-fold (14). RegB activity in vitro is very low but is enhanced by a factor up to 100 by the ribosomal protein S1 (16).
Not all GGAG sequences are cleaved equally. When located in a coding sequence, the tetranucleotide is poorly recognized or not at all. The GGAG motifs found in the SD regions of middle or late mRNAs are also generally resistant to RegB (10–14). This indicates that RegB recognizes not only the GGAG motif but also another signal. In the absence of clear sequence outside the GGAG, Lebars et al. (17) proposed the RegB to recognize a structured conformation of the GGAG sequence that is presented or stabilized by the 30S ribosomal protein S1.
Gene regB is transcribed from a typical early promoter, immediately upstream of the gene. The expression of regB gene seems to be regulated autogenously by attacking its own mRNA within GGAG motif in the SD region and at three additional GGAG motifs within its coding sequence (11). The availability of T4 regB deletion mutant reveals that the gene is non-essential under standard laboratory conditions (11,18,19). However, PCR analysis showed the presence of gene regB in the genomes of four other T4-related phages (20).
More than 150 bacteriophages with morphologies similar to T4 have been described (21,22). Genomic hybridization and PCR analysis revealed that the T4-related phages vary considerably in their genomic organization (20,23–26). On the basis of the sequence comparison of the major structural genes, capsid gene (gene 23), tail sheath gene (gene 18) and tail tube gene (gene 19), four subgroups of the T4-related phages have been distinguished: the T-evens, pseudo T-evens, schizo T-evens and exo T-evens (26–28). The term ‘T4-related’ refers to all of them.
The vast majority of the known T4-related phages were isolated on enterobacteria, primarily E.coli, Shigella and Klebsiella, and most of them belong to the T-even subgroup. Among the T-even phages, the nucleotide sequences of homologous genes typically differ from each other by <5% (23,24,26). Due to their close relationship to T4, the T-even genomes can usually be analyzed and sequenced using PCR primers based on the T4 sequence (20,26). The members of the pseudo T-even subgroup are more diverse in their host range than the T-even phages, and their genomes are phylogenetically distant from T4. About 70% of the genome of the best-characterized pseudo T-even phage RB49 encodes protein homologues of T4 genes with the levels of amino acid identity ranging between 50% and 70% (28). Among the various identified RB49 genes, those encoding structural proteins have the highest levels of homology to their T4 counterparts (25,26).
In this study, we determined the regB sequences of 35 T4-related bacteriophages. We found that the regB gene of phages RB69, TuIa and RB49 diverged considerably from its T4 counterpart. Differences in the primary structure of rather a small protein with molecular mass of 18 kDa raised up a question if the endoribonuclease RegB of RB69, TuIa and RB49 retained the same unique specificity as the T4 RegB. Therefore, we examined the functional properties of RegB homologues encoded by these T4-related bacteriophages.
MATERIALS AND METHODS
Bacterial and bacteriophage strains
E.coli strain BE (sup0) was a gift from Dr L. W. Black. E.coli strain CR63 (supD, ser) was kindly provided by Dr K. N. Kreuzer. E.coli RNase E+ and RNAse E– isogenic strains N3433 (genotype HfrH, lacZ43, lambda–, relA1, spoT1, thi-1) and N3431 were kindly supplied by Dr P. Régnier. E.coli JM101 (Amersham Biosciences) was used for transformation and preparation of plasmid DNA. E.coli C41(DE3), a derivative of E.coli BL21(DE3) F– dcm ompT hsdS(rB–mB–) gal (DE3) (Novagen) allowing overexpression of proteins at an elevated level without toxic effect (29), was used in plasmid–phage assays.
Phages T2, T6, M1, K3, Ox2, TuIa and TuIb were obtained from Dr U. Henning. Phages RB2, RB3, RB14, RB15, RB23, RB27, RB32, RB33, RB69, RB70, KC69, Pol, LZ1, LZ4, LZ5, LZ6, LZ7, LZ9, LZ10, U4 and U5 were kindly provided by Dr K. Carlson. Bacteriophages T4D wild-type, RB6, RB10, RB26, RB42, RB49, RB51, RB61 and RB62 were kindly supplied by Dr W. B. Wood. Phage T4 regB– (regBL52) was a gift from Dr M. Uzan. All phages were grown in E.coli BE (sup0), except for TuIb grown in E.coli CR63 (supD, ser).
PCR and sequencing procedures
Gene regB of the T4-related bacteriophages was amplified by PCR using the specific primers. Phage plaques were used as a source of DNA templates for PCR amplification. The PCR protocol involved 30 cycles with a program of denaturation at 92°C for 1 min, primer annealing at 50°C for 1 min and extension at 72°C for 1 min. In some cases, the annealing temperatures had to be adapted for some of these templates. For example, an RB69 PCR product was obtained at an annealing temperature of 48°C.
Sequencing was performed using a CycleReaderTM DNA sequencing kit (Fermentas AB). The DNA template for the sequencing reactions was in the form of a PCR fragment or in the form of a purified genomic phage DNA prepared for the direct sequencing as described by Kricker (30). The oligonucleotide primers were 5' end-labeled by T4 polynucleotide kinase (Fermentas AB) with ATP or ATP (Amersham Biosciences). Phage-specific primers for PCR and sequencing procedures were used as follows.
T4-specific primers
To get the PCR-generated DNA fragments containing gene regB of T4-related bacteriophages, four oligonucleotide primers were synthesized according to the known phage T4 sequences: Pr. 1, 5'-GCTTCGTCGTCACTTTGAGTCGG, 39–60 nt of the T4 regB gene; Pr. 2, 5'-GAAGCCTTTCCTGGTAAACGACG, complementary to 415–437 nt of T4 gene regB; Pr. 3, 5'-GAGGTTCGTGCCGAAGTTTCAAAGTC, 223–248 nt of T4 gene vs.3; Pr. 4, 5'-GGACATTACTGAAAGTATGCTCGGAGC, complementary to 53–79 nt of T4 vs.1 gene. Additional T4-specific primers 5'-GAATTCATTAACTTCTAAAACATG, 5'-GACTCCTGCTGCTTTTGATGCCTC, 5'-GAGGCATCAAAAGCAGCAGG, 5'-GACCATGTTTTAGAAGTTAATGA, 5'-GAGGAGAATAACATGACTATCAA, 5'-GTTTACTTTTCCTCTTGACTGTGGTA, 5'-ACAGTTATTCTTTAAATCTAATC, 5'-GTCTTGATGACTTCCAGGAAGTAGTCC were used to sequence the genomic region with gene regB in 32 T4-related phages. A pair of the T4 gene vs.1-specific primers, 5'-CAATGAGGTAAGCATGAGAAAAGCAC and 5'-ACTCCGCCAAAGCTTTCTTGCC, was used to get a DNA fragment with gene vs.1 of RB69.
RB69 (TuIa)-specific primers
Primer 5'-ATACGCATGGTGACCTTTCTTATC, complementary to nucleotides 232–255 of gene vs.1 was synthesized according to the obtained sequence of RB69 gene vs.1. This primer together with the T4-specific primers mentioned above and the RB69-specific primers 5'-GCCTGGTAGTCCAAGAGACTTTGCAGC, 5'-GGTTTAGTTGCTCAGGCGAGCA were used to sequence the gene regB as well as the upstream genes vs.3 and vs.4 of bacteriophages RB69 and TuIa.
RB49-specific primers
Based on the sequence of pseudo T-even phage RB49 gene vs.1, which was available in GenBank (31), we designed primer 5'-GAATTGCTCGCCGTATTGATAGGC, complementary to nucleotides 136–159 of gene vs.1 of RB49. This primer and other RB49-specific primers 5'-GCCCTATCACCAGAAAGTCAAGC, 5'-GATTAGATCTACTGTCATCCCG, 5'-GGTCGGTCTTCCATTTTCAGGAAGTCG, 5'-GATATGACGATCGGTTAGACGG, 5'-GATCTTCGCATTGGTTTTGCGC, 5'-GTGAACAACCGTCTGGATCTAGG, 5'-GTGAGCGAGAAACTCTTATTAACC, 5'-GCACATAATTCGACCTAGCGTACC were used to determine the sequence of RB49 gene regB and the sequence of the upstream gene regB.1 as well.
RB42-specific primers
Using the RB69 gene vs.1-specific primer 5'-ATACGCATGGTGACCTTTCTTATC and the genomic DNA of pseudo T-even phage RB42, we sequenced the proximal part of the RB42 gene vs.1. Based on this sequence, we designed the RB42-specific primer 5'-GCATCATACAAAGCAACCAGGTC, which was used to sequence the gene vs.1 upstream region of phage RB42.
Gene sequences
The nucleotide sequences have been submitted to the EMBL/GenBank database under the accession numbers: AJ488513 (for phage T2), AJ488518 (T6), AJ488514 (M1), AJ488515 (K3), AJ488516 (Ox2), AJ490327 (TuIa), AJ488517 (TuIb), AJ606891 (Pol), AJ488519 (RB2), AJ488520 (RB3), AJ488521 (RB6), AJ488522 (RB10), AJ488523 (RB14), AJ488524 (RB15), AJ488525 (RB23), AJ488526 (RB26), AJ488527 (RB27), AJ606901 (RB32), AJ606902 (RB33), AJ496183 (RB49), AJ606903 (RB51), AJ606904 (RB61), AJ606905 (RB62), AJ439451 (RB69), AJ606889 (RB70), AJ606890 (KC69), AJ606892 (LZ1), AJ606893 (LZ4), AJ606894 (LZ5), AJ606895 (LZ6), AJ606896 (LZ7), AJ606897 (LZ9), AJ606898 (LZ10), AJ606899 (U4) and AJ606900 (U5).
Plasmid constructions
Plasmid carrying the DNA fragment with RB49 gene vs.1 upstream sequence
The 109 bp RB49 DNA fragment, starting 37 bases upstream of the gene vs.1 initiation codon ATG and ending 72 bases downstream of ATG, was amplified using two oligo primes: a direct primer, 5'-TATGGATCCAGAAAGTTAAGCCAAACAGG, which contains BamHI restriction site, and a reverse primer, 5'-GCTAGTCGACGCGTGTGTAGC, containing SalI restriction site. The amplified DNA fragment was then digested with restriction enzymes and inserted into the BamHI and SalI sites of pET21(+) (Novagen) to produce plasmid pAZRB49vs.1', carrying truncated RB49 gene vs.1. The cloned sequence was confirmed by the dideoxy chain termination method (32).
Plasmids carrying the DNA fragment with gene regB
The DNA fragments containing the gene regB together with its SD sequence of phages T4, RB69 and RB49 were cloned into plasmid vector pET21(+) (Novagen) resulting in three recombinant plasmids pLPT4regB, pLTRB69regB and pLPRB49regB, respectively.
Plasmid pLPT4regB carrying gene regB was constructed by inserting a 0.49 kb DNA fragment encoding regB gene of phage T4 between the Ecl136II and XhoI restriction sites of plasmid pET21(+). The T4 DNA fragment carrying regB gene was generated by PCR using two primers: a direct primer, 5'-CAGTTAAGAGGAGAATAACATGAC and a reverse primer, 5'-GTGCTTTTCTCGAGCTTACCTCATTG, containing XhoI restriction site. The obtained DNA fragment with gene regB was treated with T4 DNA polymerase and digested with XhoI afterwards.
The recombinant plasmid pLTRB69regB was constructed as follows. The 1.3 kb DNA fragment was amplified from phage RB69 genome using two specific oligo primers: a direct primer, 5'-GGTTTAGTTGCTCAGGCGAGCA and a reverse primer, 5'-ATACGCATGGTGACCTTTCTTATC. The amplified DNA fragment was then digested with restriction endonucleases HincII and XbaI. The resulted 0.48 kb restriction fragment carrying the regB gene of phage RB69 was filled in, and the blunt-ended DNA fragment was inserted into the SacI site of plasmid vector pET21(+).
Plasmid pLPRB49regB was constructed as follows. A direct primer, 5'-CCGTTTGTATACTGAATTCGCAATTTG carrying EcoRI restriction site and a reverse primer, 5'-GCCCTATCTCGAGAAAGTCAAGC carrying XhoI restriction site were used to amplify a DNA fragment with gene regB of phage RB49. The amplified DNA fragment was then digested with restriction enzymes and the resulting 0.49 kb fragment was cloned into EcoRI–XhoI sites of plasmid vector pET21(+).
The nucleotide sequences of the cloned DNA fragments were confirmed by the dideoxy chain termination method (32). Standard procedures for the isolation and manipulation of plasmid DNA, and for the construction and identification of recombinant plasmids were used throughout (33). DNA restriction endonucleases, T4 DNA ligase and polymerase, DNA polymerase I (Klenow fragment), Taq DNA polymerase, Pfu DNA polymerase and CycleReaderTM DNA sequencing kit were obtained from Fermentas AB.
Assays of RegB endoribonuclease activity in phage-infected cells
To detect the RegB cleavage sites in the phage-induced transcripts, the E.coli BE cells were grown at 30°C to density 3 x 108 cells/ml in Luria–Bertani (LB) medium and then were infected with phages T4, RB69, RB49 or TuIa in the presence or in the absence of chloramphenicol. Chloramphenicol (150 μg/ml) was added 1 min prior to phage infection. The cells were collected 5 min after infection, lysed immediately and total cellular RNA was purified. Cleavages were analyzed by primer extension sequencing.
Assays of RegB endoribonuclease activity in plasmid–phage systems
The activities of RegB endoribonucleases of different phages were tested on transcripts induced either by phage infection or from recombinant plasmids containing phage genes. To test the activity of plasmid-encoded RegB endoribonucleases on phage-induced transcripts, the E.coli C41(DE3) cells harboring the recombinant plasmids pLPT4regB, pLTRB69regB or pLPRB49regB were grown in LB medium supplemented with ampicillin (40 μg/ml) at 30°C to A600 = 0.6. RegB was induced by the addition of isopropyl-?-D-thiogalactopyranoside (IPTG) to 1 mM, and 15 min later, the cells were infected either by phage RB49 or T4regBL52 (m.o.i. = 10). The cells were withdrawn at 4 or 5 min after infection, lysed immediately and total cellular RNA for primer extension sequencing was purified.
The activities of RegB endoribonucleases of different phages were also tested in reverse experiment. For this, E.coli C41(DE3) cells harboring recombinant plasmid pAZRB49vs.1' were grown in LB medium supplemented with ampicillin (40 μg/ml) at 30°C to A600 = 0.6. The RNA to be tested was induced from plasmid by the addition of IPTG to 1 mM, and 30 min later, the cells were infected by phages T4, RB69, TuIa, RB49 or RB42 (m.o.i. = 10). The cells were withdrawn at 5 min after infection, lysed immediately and total cellular RNA was purified. Cleavages were analyzed by primer extension sequencing.
RNase E cleavage assay
E.coli strains N3433 (rne+) and N3431 (rne–) were grown in LB medium supplemented with thymine (50 μg/ml) at 30°C to A600 = 0.5. With or without shifting of the cultures to 43°C for 30 min, cells were infected with T4D wild-type (m.o.i. = 10). The cells were withdrawn at 5 min after infection, lysed immediately and total cellular RNA was purified. RNase E cleavage was determined by primer extension sequencing.
RNA preparation and primer extension analysis of phage mRNAs
Total RNA from phage-infected E.coli cells was phenol extracted and used for the primer extension analysis or RNA sequencing under conditions of primer excess, using avian myeloblastosis virus reverse transcriptase, as described in (10,12). For details see Truncaite et al. (34). A total of eight synthetic oligonucleotides complementary to the coding sequences of genes of phages T4, RB69 (TuIa), RB49 were used to prime reverse transcriptase: Pr. T4regB(R1), 5'-GCGCGATCAAGAAGATGTTGAG, complementary to nucleotides 137–158 of the T4 regB gene; Pr. T4regB(R2), 5'-GCGGAGGCATACTCAGGAATTC, complementary to nucleotides 235–256 of the T4 regB gene; Pr. RB69regB(R1), 5'-GGCACGATCTAAGAGATGTTGAG, complementary to nucleotides 137–159 of the RB69 regB gene; Pr. RB49regB.1(R1), 5'-GCACATAATTCGACCTAGCGTACC, complementary to nucleotides 91–114 of the RB49 regB.1 gene; Pr. RB49regB(R1), 5'-GATCTTCGCATTGGTTTTGCGC, complementary to nucleotides 63–84 of the RB49 regB gene; Pr. RB49regB(R2), 5'-GCCCTATCACCAGAAAGTCAAGC, complementary to nucleotide 23–45 downstream of the termination codon of the RB49 regB gene; Pr. RB49vs.1(R1), 5'-GAATTGCTCGCCGTATTGATAGGC, complementary to nucleotides 136–159 of the RB49 vs.1 gene; Pr. RB49g43(R1), 5'-GTTGGCGTATATTCAACGTAACGCTC, complementary to nucleotides 76–101 of the RB49 gene 43.
RESULTS AND DISCUSSION
Sequences of gene regB of T4-related phages
The RegB endoribonuclease of bacteriophage T4 presents several unique aspects. It is the first description of the virus-encoded endoribonuclease that participates in the control of the expression of a number of phage early genes (10). This enzyme has almost absolute sequence specificity towards the GGAG tetranucleotides located in the SD regions of early mRNAs (14). Finally, RegB has a unique primary structure that presents no homology to other proteins, in particular to the other known RNases (35). The previous PCR analysis has shown the presence of regB homologues in the genomes of four T4-related phages, K3, RB70, T2 and T6 (20), but the nucleotide sequences have not been determined. In this work, we analyzed the conservation level of the gene regB in a wide group of T4 relatives. For this, we performed PCR and sequencing analysis of the genomic regions carrying gene regB of 36 T4-related phages.
A pair of primers based on the T4 gene regB sequence, Pr. 1–Pr. 2, and a pair of primers flanking the T4 gene regB, Pr. 3–Pr. 4, were used to amplify the analogues of gene regB of 36 T4-type phages. Using these primers, we obtained the PCR products of the expected sizes of 32 T4-related phages. The obtained PCR fragments were sequenced. The four phages, RB69, TuIa, RB42 and RB49, did not yield the PCR fragments with the primers used. A pair of primers based on the T4 gene vs.1 sequence yielded PCR fragment of RB69, which was sequenced and shown to contain gene vs.1 homologue. Additional primers were designed based on the newly obtained sequences of RB69 (see Materials and Methods). Using these primers, we were able to determine the sequence of gene regB of phages RB69 and TuIa. The sequence of gene vs.1 of pseudo T-even phage RB49 was available in GenBank (31). Based on this sequence, we designed primer and sequenced the upstream region of vs.1 of RB49. The RB49-specific primers were designed, and we defined the complete nucleotide sequence of gene regB of phage RB49. However, our attempts to obtain the sequence of gene regB of phage RB42 failed.
The sequences of the regB genes in 32 T4-related phages appeared to be very similar to each other. The deduced primary structures of RegB endoribonucleases of these phages were almost identical (98.6–100%) to the T4 RegB (Figure 1). Based on PCR analysis of generally conserved regions, most of these phages have been previously classified as T-evens (20). The phages RB69 and TuIa were also considered as the T-evens (26,28), but the DNA hybridization studies and sequence analysis of some of the genes indicated these phages could occupy an ‘intermediate’ position between T-even and pseudo T-even phages (26,36). The term ‘mezzo T-evens’ has been proposed to describe such a class of ‘intermediate’ T-even type phages (H. Krisch, personal communication). We also found that the RegB protein of RB69 and TuIa differed from the T4 RegB at a higher level that would be expected for T-evens: the primary structures of RegB endoribonucleases of phages TuIa and RB69 differed from the T4 RegB protein by 22.5%, but were identical to each other (Figure 1). The RegB of the pseudo T-even phage RB49 showed only 43.0% sequence identity with its T4 homologue and diverged mostly from the rest of the phages examined (Figure 1).
Figure 1. Amino acid sequence alignment of RegB endoribonucleases of T4-related phages. The RegB sequences of the phages were aligned with the T4 RegB sequence using the ClustalW program. A white background indicates amino acid motifs common to all of the T4-related subgroups. The sequences shown with black backgrounds are not well conserved. A dash indicates a space, which was inserted in the sequence to preserve the alignment. An asterisk (*) means that the residues in the column are identical; a double dot (:) indicates the conserved substitutions; a dot (.) indicates the semi-conserved substitutions.
It should be noted that we could not detect the regB gene in the genome of pseudo T-even phage RB42. The open reading frame (ORF) that we had sequenced upstream of the gene vs.1 of phage RB42 showed no homology to the T4 gene regB. Plasmid-induced transcripts carrying the GGAG motifs that were known to be well processed by T4 RegB were not cleaved after infection with RB42 (data not shown). This strongly suggested that the phage RB42 could lack the gene regB homologue. In support, it was recently shown that regB gene was also absent in the genome of phage KVP40, which is more distantly related to the T-evens (37). The maintenance of regB gene in most of the closely related T4-like phages might have been associated with a requirement for gpRegB in certain natural conditions that are not shared by phages distantly related to the T4.
Transcriptional and post-transcriptional control of regB expression in T4-related bacteriophages
Figure 2 shows the genes and the sites for transcriptional and post-transcriptional regulation in the regB region of four T4-type phages. In T4, the transcript for gene regB is initiated from a typical early promoter, PEregB, immediately upstream the gene (11). We analyzed the nucleotide sequence of the 5' flanking region of gene regB in 34 T4-related phages and found that all these phages possessed the promoter sequences immediately upstream the gene regB. The –35 and –10 promoter elements of PEregB of 32 T-even phages were identical to those of T4, and remarkably matched the T4 early promoter consensus elements –35 GTTTAC(a/ttt) and –10 TA(t/c)(a/t)AT spaced by 16–17 bp (2,38–41). The promoter sequences for gene regB of phages RB69 and TuIa also shared features of the T4 early promoters (Figure 3C). Thus, the regulation of regB transcription appeared to be similar in 34 phages tested.
Figure 2. Genes and promoters in the 1.10 kb genomic region with gene regB of bacteriophages T4, RB69 (TuIa) and RB49. The locations of early promoters are shown, as well as the positions of potential RegB cleavage sites within the SD sequences and in the coding sequences of regB. Vertical arrows show the sites susceptible to the RegB cleavage.
Figure 3. Primer extension analyses of transcripts for gene regB of phages T4, RB69 and TuIa. (A) Primer extension sequencing reaction of RNA isolated from E.coli BE cells at 5 min post-infection with bacteriophage T4 at 30°C. Primer extension reactions of RNA isolated at 1–15 min post-infection from the cells that were infected with phage T4 at 30°C are shown next to the sequencing reactions. (B) Primer extension sequencing reactions of RNA isolated from E.coli BE cells at 5 min post-infection with either phage RB69 or TuIa at 30°C. Primer extension sequencing reactions were performed using the primers Pr. T4regB(R1) (A) and Pr. RB69regB(R1) (B). The sequencing lanes are labeled with the dideoxynucleotides used in the sequencing reactions. The time (minutes) of post-infection that each RNA was isolated is noted at the top of the figures. The initiating nucleotides for the transcripts, the GGAG motifs within the SD sequences, the initiating codons, as well as the GGAG motifs in the coding region of gene regB mRNA are noted. (C) The nucleotide sequences of the 5' flanking region of gene regB of phages T4, RB69 and TuIa. The –35 and –10 regions of the early promoters, the initiating nucleotides for the transcripts, the GGAG motifs within the SD sequences, the initiation codons, as well as the GGAG motifs in coding regions of genes, are shown with black backgrounds. Vertical arrows denote the positions of RegB cleavage. Convergent arrows indicate inverted repeats. Asterisks indicate the termination codons for the upstream genes.
In RB49 genome, no T4 consensus sequences of either early or middle mode promoters were found (28,31). Analysis of the transcription of several regions of the RB49 genome, such as the 5' flanking regions of genes 43 and 32 (28,31), revealed the early promoters of RB49 to contain the sequences matching E.coli 70-dependent promoter sequence –35 cTTGACa and –10 TATAAT spaced by 16–18 bp (42–45). However, we could not detect any typical promoter sequence just upstream the gene regB, while the primer extension analyses of regB transcripts showed a weak transcription start site for this gene (Figures 4A and 5B). According to this site, we deduced promoter, PEregB, which contained the –35 sequence GTTTAAA and the –10 sequence TACTGG spaced by 15 bp (Figure 5). Thus, PEregB slightly matched the –35 element of T4 early promoters but had a poor –10 element, and the spacing region between them was too short in comparison to the T4 early promoters (2,38–41). Nevertheless, the in vitro transcription experiments demonstrated that the E.coli RNA polymerase could recognize this sequence on the PCR-generated DNA fragment and initiate transcription at the same position as in vivo (data not shown). Primer extension analysis of RB49 regB transcription showed that the PEregB acted as a very weak promoter (Figures 4A and 5B), and regB appeared to be transcribed mostly into the polycistronic transcripts from the more distal promoter PEregB.1 (Figure 2), which had a typical E.coli 70-dependent promoter sequence and was much more effective (Figure 5).
Figure 4. Primer extension analyses of phage RB49 transcript for gene regB. (A and B) Primer extension sequencing reactions of RNA isolated from E.coli BE cells at 5 min post-infection with RB49 at 30°C. Primer extension reactions of RNA isolated at 1–15 min post-infection from cells that were infected with RB49 at 30°C are shown next to the sequencing reactions. The sequencing lanes are labeled with dideoxynucleotides used in the sequencing reactions. The time (minutes) of post-infection that each RNA was isolated is noted at the top of the figures. (C) Primer extension sequencing of RNA isolated from E.coli N3433 (rne+) and N3431 (rne–) cells at 5 min post-infection with RB49 at 30 and 43°C. The GGAU motif within the SD sequence, the initiation codon for regB gene, the initiating nucleotide for the RegB-dependent transcript, as well as the 5' end nucleotide of RNase E processed transcript are noted. Primer extension sequencing reactions were performed using primers Pr. RB49regB(R1) (A) and Pr. RB49regB(R2) (B and C). (D) Nucleotide sequence flanking the RNase E cleavage site of the gene regB transcript and the potential secondary structures close to the cleavage site are shown. The arrow indicates the position of the RNase E cleavage. The 5' end nucleotide of RNase E processed transcript is shown with a black background. Asterisks indicate the termination codon for gene regB.
Figure 5. Primer extension sequencing of the transcripts for genes regB.1 (A), regB (B), vs.1 (C) and 43 (D) of phage RB49. Primer extension sequencing reactions of RNA isolated at 5 min post-infection from E.coli BE cells that were infected with RB49 in the absence or in the presence of chloramphenicol at 30°C. Sequencing of DNA fragment carrying gene 43 of phage RB49 is also presented (D). The initiating nucleotides for the transcripts, the potential RegB cleavage sites within the SD sequences, as well as initiation codons for the corresponding genes are noted. Convergent arrow indicates inverted repeat sequences. Primer extension reactions were performed using primers Pr. RB49regB.1(R1) (A), Pr. RB49regB(R1) (B), Pr. RB49vs.1(R1) (C) and Pr. RB49g43(R1) (D). The nucleotide sequences of the 5' flanking region of genes regB.1, regB, vs.1 and 43 of phage RB49 are shown at the bottom of the figure. The –35 and –10 regions of the early promoters, the initiating nucleotides for the transcripts, the GGAG and GGAU motifs within the SD sequences, as well as initiation codons are shown with black backgrounds. Vertical arrows denote the position of RegB cleavage. Convergent arrows indicate inverted repeats. Asterisks indicate the termination codons for the upstream genes.
A characteristic property of ribonucleases is the ability to autoregulate their synthesis in the cell. In E.coli, the genes for RNase III (46–48), RNase E (49–51), RNaseII (52,53) and PNPase (54–56) are known to be autoregulated or interregulated by controlling the degradation rate of their own mRNAs. Previous studies showed that the T4 endoribonuclease RegB efficiently cleaved its own mRNA within the GGAG motif located in the SD region and, less efficiently, the three additional GGAG motifs located in the coding sequence of the transcript for gene regB (11). Thirty-two T-evens analyzed in this study also carried the GGAG motif in the SD region and contained three or at least two GGAG motifs within the proximal part of the coding sequence of gene regB. Phages RB69 and TuIa contained the GGAG motif in the SD region and only one GGAG downstream from the initiation codon AUG (Figure 2). The SD region of RB49 gene regB contained a motif GGAU. In T4, only two exceptions were found where the RegB cleaved a GGAU sequence (13). The coding sequence of RB49 regB mRNA lacked the putative RegB cleavage sites at the positions observed for T4, but had two GGAU motifs and the GGAG motif within the distal part of the coding sequence of regB transcript (Figure 2). Thus, autoregulation of expression seemed to be natural to the phages studied, although the concentration of the putative RegB target sites within the regB transcripts was different.
We tested the RegB nucleases of phages RB69, TuIa and RB49, that exhibited higher degree of divergence from T4, for their activity towards GGAG and GGAU motifs located in the SD regions and in the coding sequences of their regB transcripts. Figure 3A and B shows the results of primer extension sequencing on RNAs extracted from the cells after infection with T4, RB69 and TuIa. Two 5' truncated RNA termini were detected in every case. The first cut occurred within the SD sequence and the second one laid in the coding sequence downstream from AUG codon in the cases of RB69 and TuIa, as well as in T4.
Figure 4A shows the primer extension sequencing and the kinetics of accumulation of RB49 regB mRNAs. The 5' end of mRNA assigned to the transcription initiation appeared as soon as the first minute of infection, while the 5' end of mRNA cleaved at the middle of the GGAU motif of the SD region was detected at the second minute of infection. Primer extension sequencing of the RB49 mRNAs for gene regB extracted from chloramphenicol-treated cells indicated that the cleavage was phage induced (Figure 5B). Thus, we can conclude that autoregulation of the expression at the post-transcriptional level is common to the regB of phages RB69, TuIa and RB49.
Two GGAU motifs as well as the GGAG located in the coding sequence of RB49 regB were resistant to RB49 RegB nucleolytic activity (data not shown). While seeking for the RegB-dependent cleavages in the coding sequence of the transcript for RB49 gene regB, the additional nucleolytic cut was observed within the distal part of the mRNA (Figure 4B). Primer extension analysis showed that the 5' end of the truncated RB49 regB mRNA appeared at 3 min of infection and continued to accumulate onward. The 5' end of truncated RNA was seen even when RB49 infection was carried out in the presence of chloramphenicol (data not shown) indicating that this endonucleolytic cleavage was caused by a host-encoded endoribonuclease. The sequence where the cut occurred matched the processing site for the E.coli RNase E (3,23,57–60): the region was A–U rich and three stable stem–loops were supposed adjacent (Figure 4D).
To test whether the truncated mRNA could be the product of E.coli RNase E cleavage, total RNA was extracted from isogenic E.coli wild-type and rne (ts) strains after infection with phage RB49. The extracted mRNA was analyzed by primer extension sequencing. In the wild-type background, the truncated mRNA of RB49 regB was detected either in 30°C or in 43°C. In the rne (ts) background at 43°C, the 5' ends of truncated mRNA were not observed, indicating that the 5' ends were generated by RNase E-dependent cleavage and not by any other endonucleolytic event (Figure 4C). Therefore, we can conclude that the RegB of RB49 controls its biosynthesis by attacking its own mRNA within GGAU motif in the SD region, but in addition, the E.coli ribonuclease E is utilized for the degradation of regB transcripts of RB49. We looked for the RNase E processing sites in the regB transcripts of phages T4 and RB69, but the systematic primer extension analysis did not reveal RNase E-dependent cleavages.
E.coli RNase E was originally implicated in processing of T4 transcripts for genes 32 (57,58) and -gt (23). Furthermore, this ribonuclease was shown to have a major role in the degradation of many T4 messages (9). The RNase E-dependent processing sites in the gene 32-transcription unit was shown to be conserved in six T4-related phages (23,61). In spite of the sequence heterogeneity in this region, RNase E cleaved mRNAs for gene 32 of these phages (23). However, in case of phage RB49 infection, unlike that of T4, the host RNase E was shown to have no major role in processing the gene 32-transcription unit (31). Therefore, we present perhaps the first clear evidence of the E.coli RNase E activity towards phage RB49 mRNAs.
Specificities of different RegB endoribonucleases
The RegB nuclease of bacteriophage T4 shows high specificity to the GGAG sequence but exhibits different activity in vivo towards different classes of mRNA (14). The GGAG-carrying RNAs that are uncut during T4 infection are also resistant to RegB in the two-plasmid systems in vitro indicating that the information identifying the substrate for RegB is carried in cis by RNA molecule (14,17,62). However, clear sequence motif outside GGAG distinguishing substrates and non-substrates of RegB has not been identified. It was shown that RegB preferentially cleaves GGAG motifs involved in a particular secondary structure, the formation of which is favored by the ribosomal protein S1 (17,63).
However, little information is available in the literature concerning the protein determinants of RegB that contribute to the recognition specificity and cleavage activity of this enzyme. Examination of the T4 regB mutants constructed by a site-directed mutagenesis revealed the potent catalytic residues His-48 and His-68 (35,64). The Arg-52 was proposed to be important for the binding of the substrate RNA. The comparison of the primary structures of 35 RegB ribonucleases determined in this study showed that all these three residues were conserved between the 35 T4-related phages tested, except for RB49 RegB, where histidine H68 was changed to asparagine (Figure 1). Moreover, the RegB of the more distant T4 relatives had multiple variations in primary structure of the protein, and the sequence of RB49 RegB was shorter than the RegB of T4 by 13 amino acids. Therefore, we wondered if the RegB endoribonucleases encoded by distant T4-like phages retained the same functional properties as the T4 RegB.
As it was already mentioned, we found that the RegB endonucleases of RB69 and TuIa cleaved the GGAG motifs within the SD regions and in the coding sequences of their mRNAs for gene regB. The RegB of RB49 processed only the GGAU motif located within the ribosome-binding region of its regB mRNA but not the GGAU or GGAG motifs located within the coding region. In order to test the ability of RB49 RegB to cleave the GGAG motifs, we performed primer extension sequencing of RB49 transcripts for genes regB.1 and vs.1 (Figure 2), as well as for gene 43 (31), that were known to carry GGAG motifs in their SD regions. The mRNA for gene regB.1 was processed within SD sequence by ribonuclease RegB of RB49 (Figure 5A), but the mRNAs of genes vs.1 and 43 were resistant (Figure 5C and D). To ensure that the observed cuts in the SD sequences of genes regB.1 and regB were caused by endoribonuclease RegB and not by E.coli endonucleolytic activity, the primer extension sequencing experiments were also carried on RNAs extracted from the cells after infection with RB49 in the presence of chloramphenicol. The 5' end of truncated mRNA did not appear in both cases (Figure 5A and B) indicating that the activity was phage induced.
The noticeable selectivity against the GGAG and GGAU motifs observed for RB49 RegB might depend on the differences in the primary structure of this enzyme. To test this, we examined the capability of RegB of phages T4, RB69 and TuIa to cleave those GGAG-carrying mRNAs that were not cleaved by RB49 RegB. For this, we cloned the proximal part of gene vs.1 of phage RB49 into an inducible plasmid pET21(+) and transformed E.coli C41(DE3) cells with the resulted plasmid pAZRB49vs.1'. The transcripts for the proximal part of RB49 gene vs.1 were induced by addition of IPTG to the medium, and 15 min later, the cells were infected with one of the four phages. Total RNA was extracted, and the RegB cleavage sites were mapped by RNA sequencing of plasmid-induced transcripts. During T4, RB69 or TuIa infection, the RB49 vs.1 transcript induced from plasmid was cleaved in the SD region, though the efficiency of T4 RegB cleavage was much lower than those of RB69 and TuIa (Figure 6A). As expected, the plasmid-induced RB49 vs.1 transcript stayed resistant during infection of RB49. The RegB-dependent processing event was also not observed after infection with phage RB42 (Figure 6A).
Figure 6. Susceptibility of RB49 transcripts for genes vs.1 and 43 to the RegB endoribonucleases of different T4-related bacteriophages. (A) Primer extension sequencing of RNA isolated from E.coli C41(DE3) cells, harboring recombinant plasmid pAZRB49vs.1', after 5 min post-infection at 30°C with one of the five phages indicated above the figure. The transcript for the gene vs.1 was induced from the plasmid by the addition of IPTG 30 min before infection. The control experiment without phage infection is also shown. Cleavages were detected by primer extension sequencing using T7 terminator primer 5'-GCTAGTTATTGCTCAGCG. (B and C) Primer extension sequencing of RNA isolated from E.coli C41(DE3) cells, harboring one of the three recombinant plasmids indicated above the figures, at 5 min post-infection with phage RB49 at 30°C. RegB endoribonuclease of phages T4, RB69 or RB49 was induced from the corresponding plasmids by the addition of IPTG 15 min before infection. The control experiment shows the primer extension sequencing of RNA isolated from E.coli C41(DE3) cells at 5 min post-infection with RB49 at 30°C. The sequencing lanes are labeled with dideoxynucleotides used in the sequencing reactions. The initiating nucleotides for the RB49 transcripts of genes vs.1 (B) and 43 (C), the GGAG motifs within the SD sequences, the initiation codons for the corresponding genes are noted. Primer extension sequencing reactions were performed using primers Pr. RB49vs.1(R1) (B) and Pr. RB49g43(R1) (C).
In the reverse experiment, we cloned the regB genes of phages T4, RB69 or RB49 into the inducible plasmid pET21(+). The RegB endoribonuclease was induced by addition of IPTG into the medium with E.coli cells carrying one of the recombinant plasmids with a cloned regB gene, and later the cells were infected with RB49. The total RNA was extracted and used for primer extension sequencing of gene vs.1 and gene 43 mRNAs of RB49. The plasmid-induced RegB endoribonucleases of T4 and RB69 cleaved the transcripts for gene vs.1 and gene 43 of RB49 at the GGAG motif present in their SD regions, while the RegB of RB49 had no effect on these mRNAs again (Figure 6B and C).
To test whether the RB49 RegB induced from the recombinant plasmid could process GGAG motifs located in the coding sequence of the T4 transcripts for gene regB, we used the E.coli C41(DE3) cells carrying the plasmid pLPRB49regB. For the control experiments, we also used E.coli C41(DE3) carrying the plasmid pET21(+) and E.coli C41(DE3) carrying the plasmids pLPT4regB or pLTRB69regB. The RegB endoribonucleases from the recombinant plasmids were induced by addition of IPTG. Later, the cells were infected with either bacteriophage T4 wild-type or the nuclease-deficient phage T4 regBL52. Total mRNA was extracted in each case of infection, and the cleavage sites were detected by primer extension sequencing. The results showed that the RB49 RegB could process the same motifs of the T4 regB transcripts as the T4 RegB but the efficiency of processing differed. The first and the third T4 RegB processing sites located in the coding sequence of regB transcript were more susceptible to the T4 or RB69 RegB ribonucleolytic attack in comparison to the second one (Figure 7A, C and D). Whereas, the RB49 RegB cleaved the second GGAG motif more efficiently than the SD, the first and the third ones (Figure 7E).
Figure 7. Susceptibility of the T4 transcripts for gene regB to the RegB endoribonuclease of phages T4, RB69 and RB49. Primer extension sequencing of mRNA for gene regB isolated from the E.coli C41(DE3) cells harboring plasmid pET21(+) at 4 min post-infection by T4 wild-type phage at 30°C (A). Primer extension sequencing of the T4regBL52 mRNA for gene regB isolated at 4 min post-infection by phage at 30°C from the E.coli C41(DE3) cells harboring plasmid pET21(+) (B) or the recombinant plasmids pLPT4regB (C), pLTRB69regB (D) and pLPRB49regB (E). Primer Pr. T4regB(R2) was used in the sequencing reactions. The sequencing lanes are labeled with the dideoxynucleotides used in the sequencing reactions. The initiating nucleotides for the transcripts, the GGAG motifs within the SD sequences, as well as the GGAG motifs in the coding region of gene regB mRNA are noted. (F) The nucleotide sequence of the 5' part of mRNA for gene regB of bacteriophage T4. The GGAG motif within the SD sequence, the initiation codon, as well as the GGAG motifs in the coding region are shown with black backgrounds. Vertical arrows denote the positions of RegB cleavage.
Taken together, the results of these experiments clearly indicate that four homologous RegB endoribonucleases encoded by phages T4, RB69, TuIa and RB49 possess different capability to recognize GGAG/U motifs flanked by the same sequences. Some GGAG-carrying transcripts that are not cleaved by the RB49 RegB can be recognized and processed by the RegB of phages T4, RB69 or TuIa though with different efficiency. Different susceptibility of the same mRNAs to the RegB endoribonucleases of more distant T4 relatives could clearly be related to the differences in the primary structure of these proteins. It seems possible to propose that the highly diverged RegB of RB49 has more tight sequence requirements for the processing site. We expect the comparative studies of the structure–function relationships between the RegB homologues may help to detect the specificity determinants carried by the RegB protein.
ACKNOWLEDGEMENTS
We thank Dr Lindsay W. Black, Dr Philippe Régnier and Dr Kenneth N. Kreuzer for E.coli strains, Dr Karin Carlson, Dr William B. Wood and Dr Marc Uzan for phages. This work was supported in part by the Lithuanian State Science and Studies Foundation.
REFERENCES
Ueno,H. and Yonesaki,T. ( (2001) ) Recognition and specific degradation of bacteriophage T4 mRNAs. Genetics, , 158, , 7–17.
Miller,E.S., Kutter,E., Mosig,G., Arisaka,F., Kunisawa,T. and Rüger,W. ( (2003) ) Bacteriophage T4 genome. Microbiol. Mol. Biol. Rev., , 67, , 86–156.
Otsuka,Y., Ueno,H. and Yonesaki,T. ( (2003) ) Escherichia coli endoribonucleases involved in cleavage of bacteriophage T4 mRNAs. J. Bacteriol., , 185, , 983–990.
Deutscher,M.P. and Li,Z. ( (2001) ) Exoribonucleases and their multiple roles in RNA metabolism. Prog. Nucleic Acid Res. Mol. Biol., , 66, , 67–105.
Zuo,Y. and Deutscher,M.P. ( (2001) ) Exoribonuclease superfamilies: structural analysis and phylogenetic distribution. Nucleic Acids Res., , 29, , 1017–1026.
Deutscher,M.P. ( (1993) ) Ribonuclease multiplicity, diversity and complexity. J. Biol. Chem., , 268, , 13011–13014.
Kushner,S.R. ( (2002) ) mRNA decay in Escherichia coli comes of age. J. Mol. Biol., , 184, , 4658–4665.
Kennel,D. ( (2002) ) Processing endoribonucleases and mRNA degradation in bacteria. J. Bacteriol., , 184, , 4645–4657.
Mudd,E.A., Carpousis,A.J. and Krisch,H.M. ( (1990) ) E.coli RNase E has a role in the decay of bacteriophage T4 mRNA. Genes Dev., , 4, , 873–881.
Uzan,M., Favre,R. and Brody,E. ( (1988) ) A nuclease that cuts specifically in the ribosome binding site of some T4 mRNAs. Proc. Natl Acad. Sci. USA, , 85, , 8895–8899.
Ruckman,J., Parma,D., Tuerk,C., Hall,D.H. and Gold,L. ( (1989) ) Identification of a T4 gene required for bacteriophage mRNA processing. New Biol., , 1, , 54–65.
Sanson,B. and Uzan,M. ( (1993) ) Dual role of the sequence-specific bacteriophage T4 endoribonuclease RegB: mRNA inactivation and mRNA destabilization. J. Mol. Biol., , 233, , 429–446.
Sanson,B. and Uzan,M. ( (1995) ) Post-transcriptional controls in bacteriophage T4: roles of the sequence-specific endoribonuclease RegB. FEMS Microbiol. Rev., , 17, , 141–150.
Sanson,B., Hu,R.-M., Troitskaya,E., Mathy,N. and Uzan,M. ( (2000) ) Endoribonuclease RegB from bacteriophage T4 is necessary for the degradation of early but not middle or late mRNAs. J. Mol. Biol., , 297, , 1063–1074.
Truncaite,L. ( (2001) ) Deletional and transcriptional analysis of the genomic region between genes 30 and 31 of bacteriophage T4. PhD Thesis, Vilnius, Lithuania.
Ruckman,J., Ringquist,S., Brody,E. and Gold,L. ( (1994) ) The bacteriophage T4 regB ribonuclease. Stimulation of the purified enzyme by ribosomal protein S1. J. Biol. Chem., , 269, , 26655–26662.
Lebars,I., Hu,R.M., Lallemand,J.Y., Uzan,M. and Bontems,F. ( (2001) ) Role of the substrate conformation and of the S1 protein in the cleavage efficiency of the T4 endoribonuclease RegB. J. Biol. Chem., , 276, , 13264–13272.
Chace,K.V. and Hall,D.H. ( (1975) ) Characterization of new regulatory mutants of bacteriophage T4. II. New class of mutants. J. Virol., , 15, , 929–945.
Valerie,K., Stevens,J., Lynch,M., Henderson,E.E. and de Riel,J.K. ( (1986) ) Nucleotide sequence and analysis of the 58.3 to 65.5-kb early region of bacteriophage T4. Nucleic Acids Res., , 14, , 8637–8654.
Repoila,F., Tétart,F., Bouet,J.Y. and Krisch,H.M. ( (1994) ) Genomic polymorphism in the T-even bacteriophages. EMBO J., , 13, , 4181–4192.
Kutter,E., Gachechiladze,K., Poglazov,A., Marusich,E., Shneider,M., Aronsson,P., Napuli,A., Porter,D. and Mesyanzhinov,V. ( (1996) ) Evolution of T4-related phages. Virus Genes, , 11, , 285–297.
Ackermann,A.W. and Krisch,H.M. ( (1997) ) A catalogue of T4-type bacteriophages. Arch. Virol., , 142, , 2329–2345.
Loayza,D., Carpousis,A.J. and Krisch,H.M. ( (1991) ) Gene 32 transcription and mRNA processing in T4-related bacteriophages. Mol. Microbiol., , 5, , 715–725.
Selick,H.E., Stormo,G.D., Dyson,R.L. and Alberts,B.M. ( (1993) ) Analysis of five presumptive protein-coding sequences clustered between the primosome genes, 41 and 61, of bacteriophages T4, T2, and T6. J. Virol., , 67, , 2305–2316.
Monod,C., Repoila,F., Kutateladze,M., Tétart,F. and Krisch,H.M. ( (1997) ) The genome of the pseudo T-even bacteriophages, a diverse group that resembles T4. J. Mol. Biol., , 267, , 237–249.
Tétart,F., Desplats,C., Kutateladze,M., Monod,C., Ackermann,H.W. and Krisch,H.M. ( (2001) ) Phylogeny of the major head and tail genes of the wide-ranging T4-type bacteriophages. J. Bacteriol., , 183, , 358–366.
Hambly,E., Tétart,F., Desplats,C., Wilson,W.H., Krisch,H.M. and Mann,N.H. ( (2001) ) A conserved genetic module that encodes the major virion components in both the coliphage T4 and the marine cyanophage S-PM2. Proc. Natl Acad. Sci. USA, , 98, , 11411–11416.
Desplats,C. and Krisch,H.M. ( (2003) ) The diversity and evolution of the T4-type bacteriophages. Res. Microbiol., , 154, , 259–267.
Miroux,B. and Walker,J.E. ( (1996) ) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol., , 260, , 289–298.
Kricker,M. ( (1994) ) Sequencing of genomic T4 DNA. In Karam,J.D. (editor-in-chief), Molecular Biology of Bacteriophage T4. ASM Press, Washington, DC, pp. 463–464.
Desplats,C., Dez,C., Tétart,F., Eleaume,H. and Krisch,H.M. ( (2002) ) Snapshot of the genome of the pseudo-T-even bacteriophage RB49. J. Bacteriol., , 184, , 2789–2804.
Sanger,F., Nicklen,S. and Coulson,A.R. ( (1977) ) DNA sequencing with chain-terminating inhibitors. Proc. Natl Acad. Sci. USA, , 74, , 5463–5467.
Sambrook,J., Fritsch,E.F. and Maniatis,T. ( (1989) ) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Truncaite,L., Pieiniene,L., Kolesinskiene,G., Zajankauskaite,A., Driukas,A., Klausa,V. and Nivinskas,R. ( (2003) ) Twelve new MotA-dependent middle promoters of bacteriophage T4: consensus sequence revised. J. Mol. Biol., , 327, , 335–346.
Sa?da,F., Uzan,M. and Bontems,F. ( (2003) ) The phage T4 restriction endoribonuclease RegB: a cyclizing enzyme that requires two histidines to be fully active. Nucleic Acids Res., , 31, , 2751–2758.
Yeh,L.S., Hsu,T. and Karam,J.D. ( (1998) ) Divergence of a DNA replication gene cluster in the T4-related bacteriophage RB69. J. Bacteriol., , 180, , 2005–2013.
Miller,E.S., Heidelberg,J.F., Eisen,J.A., Nelson,W.C., Durkin,A.S., Ciecko,A., Feldblyum,T.V., White,O., Paulsen,I.T., Nierman,W.C., Lee,J., Szczypinski,B. and Fraser,C.M. ( (2003) ) Complete genome sequence of the broad-host-range vibriophage KVP40: comparative genomics of a T4-related bacteriophage. J. Bacteriol., , 185, , 5220–5233.
Liebig,H.D. and Rüger,W. ( (1989) ) Bacteriophage T4 early promoter regions. Consensus sequences of promoters and ribosome-binding sites. J. Mol. Biol., , 208, , 517–536.
Wilkens,K. and Rüger,W. ( (1994) ) Transcription from early promoters. In Karam,J.D. (editor-in-chief), Molecular Biology of Bacteriophage T4. ASM Press, Washington, DC, pp. 132–141.
Wilkens,K. and Rüger,W. ( (1996) ) Characterization of bacteriophage T4 early promoters in vivo with a new promoter probe vector. Plasmid, , 35, , 108–120.
Sommer,N., Salniene,V., Gineikiene,E., Nivinskas,R. and Rüger,W. ( (2000) ) T4 early promoter strength probed in vivo with unribosylated and ADP-ribosylated Escherichia coli RNA polymerase: a mutation analysis. Microbiology, , 146, , 2643–2653.
Rosenberg,M. and Court,D. ( (1979) ) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu. Rev. Genet., , 13, , 319–353.
Hawley,D.K. and McClure,W.R. ( (1983) ) Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res., , 11, , 2237–2255.
Harley,C.B. and Reynolds,R.P. ( (1987) ) Analysis of E.coli promoter sequences. Nucleic Acids Res., , 15, , 2343–2361.
Lisser,S. and Margalit,H. ( (1993) ) Compilation of E.coli mRNA promoter sequences. Nucleic Acids Res., , 21, , 1507–1516.
Bardwell,J.C.A., Régnier,P., Chen,S.-M., Nakamura,Y., Grunberg-Manago,M. and Court,D.L. ( (1989) ) Autoregulation of RNase III operon by mRNA processing. EMBO J., , 8, , 3401–3407.
Matsunaga,J., Dyer,M., Simons,E.L. and Simons,R.W. ( (1996) ) Expression and regulation of the rnc and pdxJ operons of Escherichia coli. Mol. Microbiol., , 22, , 977–989.
Matsunaga,J., Simons,E.L. and Simons,R.W. ( (1997) ) Escherichia coli RNase III (rnc) autoregulation occurs independently of rnc gene translation. Mol. Microbiol., , 26, , 1125–1135.
Jain,C. and Belasco,J.G. ( (1995) ) RNase E autoregulates its synthesis by controlling the degradation rate of its own mRNA in Escherichia coli: unusual sensitivity of the rne transcript to RNase E activity. Genes Dev., , 9, , 84–96.
Diwa,A., Bricker,A.L., Jain,C. and Belasco,J.G. ( (2000) ) An evolutionarily conserved RNA stem–loop functions as a sensor that directs feedback regulation of RNase E gene expression. Genes Dev., , 14, , 1249–1260.
Ow,M.C., Liu,Q., Mohanty,B.K., Andrew,M.E., Maples,V.F. and Kushner,S.R. ( (2002) ) RNase E levels in Escherichia coli are controlled by a complex regulatory system that involves transcription of the rne gene from three promoters. Mol. Microbiol., , 43, , 159–171.
Zilh?o,R., Régnier,P. and Arraiano,C.M. ( (1995) ) The role of endonucleases in the expression of ribonuclease II in Escherichia coli. FEMS Microbiol. Lett., , 130, , 237–244.
Zilh?o,R., Cairr?o,F., Régnier,P. and Arraiano,C.M. ( (1996) ) PNPase modulates RNase II expression in Escherichia coli: implications for mRNA decay and cell metabolism. Mol. Microbiol., , 20, , 1033–1042.
Robert-LeMeur,M. and Portier,C. ( (1992) ) E.coli polynucleotide phosphorylase expression is autoregulated through an RNase III-dependent mechanism. EMBO J., , 11, , 2633–2641.
Robert-LeMeur,M. and Portier,C. ( (1994) ) Polynucleotide phosphorylase of Escherichia coli induces the degradation of its RNase III processed messenger by preventing its translation. Nucleic Acids Res., , 22, , 397–403.
Jarrige,A.C., Mathy,N. and Portier,C. ( (2001) ) PNPase autocontrols its expression by degrading a double-stranded structure in the pnp mRNA leader. EMBO J., , 20, , 6845–6855.
Mudd,E.A., Prentki,P., Belin,D. and Krisch,H.M. ( (1988) ) Processing of unstable bacteriophage T4 gene 32 mRNAs into a stable species requires Escherichia coli ribonuclease E. EMBO J., , 7, , 3601–3607.
Carpousis,A.J., Mudd,E.A. and Krisch,H.M. ( (1989) ) Transcription and messenger RNA processing upstream of bacteriophage T4 gene 32. Mol. Gen. Genet., , 219, , 39–48.
Ehretsmann,C.P., Carpousis,A.J. and Krisch,H.M. ( (1992) ) Specificity of Escherichia coli endoribonuclease RNase E: in vivo and in vitro analysis of mutants in a bacteriophage T4 mRNA processing site. Genes Dev., , 6, , 149–159.
McDowall,K.J., Lin-Chao,S. and Cohen,S.N. ( (1994) ) A + U content rather than a particular nucleotide order determines the specificity of RNase E cleavage. J. Biol. Chem., , 269, , 10790–10796.
Carpousis,A.J. and Krisch,H.M. ( (1994) ) mRNA processing and degradation. In Karam,J.D. (editor-in-chief), Molecular Biology of Bacteriophage T4. ASM Press, Washington, DC, pp. 176–181.
Jayasena,V.K., Brown,D., Shtatland,T. and Gold,L. ( (1996) ) In vitro selection of RNA specifically cleaved by bacteriophage T4 RegB endonuclease. Biochemistry, , 35, , 2349–2356.
Bisaglia,M., Laalami,S., Uzan,M. and Bontems,F. ( (2003) ) Activation of the RegB endoribonuclease by the S1 ribosomal protein is due to cooperation between the S1 four C-terminal modules in a substrate-dependent manner. J. Biol. Chem., , 278, , 15261–15271.
Sa?da,F., Odaert,B., Uzan,M. and Bontems,F. ( (2004) ) First structural investigation of the restriction ribonuclease RegB: NMR spectroscopic conditions, 13C/15N double-isotopic labelling and two-dimensional heteronuclear spectra. Protein Expr. Purif., , 34, , 158–165.(Lina Pieiniene, Lidija Truncaite, Aureli)
* To whom correspondence should be addressed. Tel: +370 5 2729146; Fax: +370 5 2729196; Email: rimasn@bchi.lt
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
+AJ439451, AJ490327, AJ496183, AJ488513–AJ488527, AJ606889–AJ606905
DDBJ/EMBL/GenBank accession nos+
ABSTRACT
The RegB endoribonuclease encoded by bacteriophage T4 is a unique sequence-specific nuclease that cleaves in the middle of GGAG or, in a few cases, GGAU tetranucleotides, preferentially those found in the Shine–Dalgarno regions of early phage mRNAs. In this study, we examined the primary structures and functional properties of RegB ribonucleases encoded by T4-related bacteriophages. We show that all but one of 36 phages tested harbor the regB gene homologues and the similar signals for transcriptional and post-transcriptional autogenous regulation of regB expression. Phage RB49 in addition to gpRegB utilizes Escherichia coli endoribonuclease E for the degradation of its transcripts for gene regB. The deduced primary structure of RegB proteins of 32 phages studied is almost identical to that of T4, while the sequences of RegB encoded by phages RB69, TuIa and RB49 show substantial divergence from their T4 counterpart. Functional studies using plasmid–phage systems indicate that RegB nucleases of phages T4, RB69, TuIa and RB49 exhibit different activity towards GGAG and GGAU motifs in the specific locations. We expect that the availability of the different phylogenetic variants of RegB may help to localize the amino acid determinants that contribute to the specificity and cleavage efficiency of this processing enzyme.
INTRODUCTION
RNases play a central role in every aspect of T4 RNA metabolism, including decay of mRNA, conversion of RNA precursors to their mature forms and end-turnover of certain RNAs. Both host and phage endo- and exoribonucleases are involved in processing of phage transcripts (1–3). A host cell contains a large number of distinct RNases approaching as many as 20 members, often with overlapping specificities (4,5). Eight distinct 3' to 5' exoribonucleases, polynucleotide phosphorylase (PNPase), RNase II, RNase D, RNase BN, RNase T, RNase PH, RNase R and oligoribonuclease (5,6), and at least five endoribonucleases, RNases III, E, G, P and I/M (6–8) have been characterized in Escherichia coli. Among these ribonucleases, RNase E has the most prominent effect on the functional and chemical decay of many T4 mRNAs, and therefore it is considered the primary endoribonuclease for phage mRNAs (9). The endoribonuclease E acts throughout all phage development, but in addition the T4 encodes its own endoribonuclease RegB, which participates in the turnover of early mRNAs of T4 (10–14).
The RegB endoribonuclease encoded by phage T4 is a sequence-specific endoribonuclease, which cleaves mRNAs in the middle of sequence GGAG or, in a few cases, in the sequence GGAU (10,11,13). The most efficient cleavages occur in the motifs located in the intergenic regions of early T4 mRNAs. Out of 25 processing sites identified (13,15), 12 occur in Shine–Dalgarno (SD) sequences in such a way making ribosome-binding site non-functional. Primarily recognized examples were observed in the SD regions of the early genes motA, frd and comC (10–12). This suggested the idea that RegB has a general role in the breakdown of T4 early mRNAs. Indeed, in the absence of RegB the chemical half-life of early transcripts of T4 is increased by 4-fold, though their functional half-life is increased slightly by <2-fold (14). RegB activity in vitro is very low but is enhanced by a factor up to 100 by the ribosomal protein S1 (16).
Not all GGAG sequences are cleaved equally. When located in a coding sequence, the tetranucleotide is poorly recognized or not at all. The GGAG motifs found in the SD regions of middle or late mRNAs are also generally resistant to RegB (10–14). This indicates that RegB recognizes not only the GGAG motif but also another signal. In the absence of clear sequence outside the GGAG, Lebars et al. (17) proposed the RegB to recognize a structured conformation of the GGAG sequence that is presented or stabilized by the 30S ribosomal protein S1.
Gene regB is transcribed from a typical early promoter, immediately upstream of the gene. The expression of regB gene seems to be regulated autogenously by attacking its own mRNA within GGAG motif in the SD region and at three additional GGAG motifs within its coding sequence (11). The availability of T4 regB deletion mutant reveals that the gene is non-essential under standard laboratory conditions (11,18,19). However, PCR analysis showed the presence of gene regB in the genomes of four other T4-related phages (20).
More than 150 bacteriophages with morphologies similar to T4 have been described (21,22). Genomic hybridization and PCR analysis revealed that the T4-related phages vary considerably in their genomic organization (20,23–26). On the basis of the sequence comparison of the major structural genes, capsid gene (gene 23), tail sheath gene (gene 18) and tail tube gene (gene 19), four subgroups of the T4-related phages have been distinguished: the T-evens, pseudo T-evens, schizo T-evens and exo T-evens (26–28). The term ‘T4-related’ refers to all of them.
The vast majority of the known T4-related phages were isolated on enterobacteria, primarily E.coli, Shigella and Klebsiella, and most of them belong to the T-even subgroup. Among the T-even phages, the nucleotide sequences of homologous genes typically differ from each other by <5% (23,24,26). Due to their close relationship to T4, the T-even genomes can usually be analyzed and sequenced using PCR primers based on the T4 sequence (20,26). The members of the pseudo T-even subgroup are more diverse in their host range than the T-even phages, and their genomes are phylogenetically distant from T4. About 70% of the genome of the best-characterized pseudo T-even phage RB49 encodes protein homologues of T4 genes with the levels of amino acid identity ranging between 50% and 70% (28). Among the various identified RB49 genes, those encoding structural proteins have the highest levels of homology to their T4 counterparts (25,26).
In this study, we determined the regB sequences of 35 T4-related bacteriophages. We found that the regB gene of phages RB69, TuIa and RB49 diverged considerably from its T4 counterpart. Differences in the primary structure of rather a small protein with molecular mass of 18 kDa raised up a question if the endoribonuclease RegB of RB69, TuIa and RB49 retained the same unique specificity as the T4 RegB. Therefore, we examined the functional properties of RegB homologues encoded by these T4-related bacteriophages.
MATERIALS AND METHODS
Bacterial and bacteriophage strains
E.coli strain BE (sup0) was a gift from Dr L. W. Black. E.coli strain CR63 (supD, ser) was kindly provided by Dr K. N. Kreuzer. E.coli RNase E+ and RNAse E– isogenic strains N3433 (genotype HfrH, lacZ43, lambda–, relA1, spoT1, thi-1) and N3431 were kindly supplied by Dr P. Régnier. E.coli JM101 (Amersham Biosciences) was used for transformation and preparation of plasmid DNA. E.coli C41(DE3), a derivative of E.coli BL21(DE3) F– dcm ompT hsdS(rB–mB–) gal (DE3) (Novagen) allowing overexpression of proteins at an elevated level without toxic effect (29), was used in plasmid–phage assays.
Phages T2, T6, M1, K3, Ox2, TuIa and TuIb were obtained from Dr U. Henning. Phages RB2, RB3, RB14, RB15, RB23, RB27, RB32, RB33, RB69, RB70, KC69, Pol, LZ1, LZ4, LZ5, LZ6, LZ7, LZ9, LZ10, U4 and U5 were kindly provided by Dr K. Carlson. Bacteriophages T4D wild-type, RB6, RB10, RB26, RB42, RB49, RB51, RB61 and RB62 were kindly supplied by Dr W. B. Wood. Phage T4 regB– (regBL52) was a gift from Dr M. Uzan. All phages were grown in E.coli BE (sup0), except for TuIb grown in E.coli CR63 (supD, ser).
PCR and sequencing procedures
Gene regB of the T4-related bacteriophages was amplified by PCR using the specific primers. Phage plaques were used as a source of DNA templates for PCR amplification. The PCR protocol involved 30 cycles with a program of denaturation at 92°C for 1 min, primer annealing at 50°C for 1 min and extension at 72°C for 1 min. In some cases, the annealing temperatures had to be adapted for some of these templates. For example, an RB69 PCR product was obtained at an annealing temperature of 48°C.
Sequencing was performed using a CycleReaderTM DNA sequencing kit (Fermentas AB). The DNA template for the sequencing reactions was in the form of a PCR fragment or in the form of a purified genomic phage DNA prepared for the direct sequencing as described by Kricker (30). The oligonucleotide primers were 5' end-labeled by T4 polynucleotide kinase (Fermentas AB) with ATP or ATP (Amersham Biosciences). Phage-specific primers for PCR and sequencing procedures were used as follows.
T4-specific primers
To get the PCR-generated DNA fragments containing gene regB of T4-related bacteriophages, four oligonucleotide primers were synthesized according to the known phage T4 sequences: Pr. 1, 5'-GCTTCGTCGTCACTTTGAGTCGG, 39–60 nt of the T4 regB gene; Pr. 2, 5'-GAAGCCTTTCCTGGTAAACGACG, complementary to 415–437 nt of T4 gene regB; Pr. 3, 5'-GAGGTTCGTGCCGAAGTTTCAAAGTC, 223–248 nt of T4 gene vs.3; Pr. 4, 5'-GGACATTACTGAAAGTATGCTCGGAGC, complementary to 53–79 nt of T4 vs.1 gene. Additional T4-specific primers 5'-GAATTCATTAACTTCTAAAACATG, 5'-GACTCCTGCTGCTTTTGATGCCTC, 5'-GAGGCATCAAAAGCAGCAGG, 5'-GACCATGTTTTAGAAGTTAATGA, 5'-GAGGAGAATAACATGACTATCAA, 5'-GTTTACTTTTCCTCTTGACTGTGGTA, 5'-ACAGTTATTCTTTAAATCTAATC, 5'-GTCTTGATGACTTCCAGGAAGTAGTCC were used to sequence the genomic region with gene regB in 32 T4-related phages. A pair of the T4 gene vs.1-specific primers, 5'-CAATGAGGTAAGCATGAGAAAAGCAC and 5'-ACTCCGCCAAAGCTTTCTTGCC, was used to get a DNA fragment with gene vs.1 of RB69.
RB69 (TuIa)-specific primers
Primer 5'-ATACGCATGGTGACCTTTCTTATC, complementary to nucleotides 232–255 of gene vs.1 was synthesized according to the obtained sequence of RB69 gene vs.1. This primer together with the T4-specific primers mentioned above and the RB69-specific primers 5'-GCCTGGTAGTCCAAGAGACTTTGCAGC, 5'-GGTTTAGTTGCTCAGGCGAGCA were used to sequence the gene regB as well as the upstream genes vs.3 and vs.4 of bacteriophages RB69 and TuIa.
RB49-specific primers
Based on the sequence of pseudo T-even phage RB49 gene vs.1, which was available in GenBank (31), we designed primer 5'-GAATTGCTCGCCGTATTGATAGGC, complementary to nucleotides 136–159 of gene vs.1 of RB49. This primer and other RB49-specific primers 5'-GCCCTATCACCAGAAAGTCAAGC, 5'-GATTAGATCTACTGTCATCCCG, 5'-GGTCGGTCTTCCATTTTCAGGAAGTCG, 5'-GATATGACGATCGGTTAGACGG, 5'-GATCTTCGCATTGGTTTTGCGC, 5'-GTGAACAACCGTCTGGATCTAGG, 5'-GTGAGCGAGAAACTCTTATTAACC, 5'-GCACATAATTCGACCTAGCGTACC were used to determine the sequence of RB49 gene regB and the sequence of the upstream gene regB.1 as well.
RB42-specific primers
Using the RB69 gene vs.1-specific primer 5'-ATACGCATGGTGACCTTTCTTATC and the genomic DNA of pseudo T-even phage RB42, we sequenced the proximal part of the RB42 gene vs.1. Based on this sequence, we designed the RB42-specific primer 5'-GCATCATACAAAGCAACCAGGTC, which was used to sequence the gene vs.1 upstream region of phage RB42.
Gene sequences
The nucleotide sequences have been submitted to the EMBL/GenBank database under the accession numbers: AJ488513 (for phage T2), AJ488518 (T6), AJ488514 (M1), AJ488515 (K3), AJ488516 (Ox2), AJ490327 (TuIa), AJ488517 (TuIb), AJ606891 (Pol), AJ488519 (RB2), AJ488520 (RB3), AJ488521 (RB6), AJ488522 (RB10), AJ488523 (RB14), AJ488524 (RB15), AJ488525 (RB23), AJ488526 (RB26), AJ488527 (RB27), AJ606901 (RB32), AJ606902 (RB33), AJ496183 (RB49), AJ606903 (RB51), AJ606904 (RB61), AJ606905 (RB62), AJ439451 (RB69), AJ606889 (RB70), AJ606890 (KC69), AJ606892 (LZ1), AJ606893 (LZ4), AJ606894 (LZ5), AJ606895 (LZ6), AJ606896 (LZ7), AJ606897 (LZ9), AJ606898 (LZ10), AJ606899 (U4) and AJ606900 (U5).
Plasmid constructions
Plasmid carrying the DNA fragment with RB49 gene vs.1 upstream sequence
The 109 bp RB49 DNA fragment, starting 37 bases upstream of the gene vs.1 initiation codon ATG and ending 72 bases downstream of ATG, was amplified using two oligo primes: a direct primer, 5'-TATGGATCCAGAAAGTTAAGCCAAACAGG, which contains BamHI restriction site, and a reverse primer, 5'-GCTAGTCGACGCGTGTGTAGC, containing SalI restriction site. The amplified DNA fragment was then digested with restriction enzymes and inserted into the BamHI and SalI sites of pET21(+) (Novagen) to produce plasmid pAZRB49vs.1', carrying truncated RB49 gene vs.1. The cloned sequence was confirmed by the dideoxy chain termination method (32).
Plasmids carrying the DNA fragment with gene regB
The DNA fragments containing the gene regB together with its SD sequence of phages T4, RB69 and RB49 were cloned into plasmid vector pET21(+) (Novagen) resulting in three recombinant plasmids pLPT4regB, pLTRB69regB and pLPRB49regB, respectively.
Plasmid pLPT4regB carrying gene regB was constructed by inserting a 0.49 kb DNA fragment encoding regB gene of phage T4 between the Ecl136II and XhoI restriction sites of plasmid pET21(+). The T4 DNA fragment carrying regB gene was generated by PCR using two primers: a direct primer, 5'-CAGTTAAGAGGAGAATAACATGAC and a reverse primer, 5'-GTGCTTTTCTCGAGCTTACCTCATTG, containing XhoI restriction site. The obtained DNA fragment with gene regB was treated with T4 DNA polymerase and digested with XhoI afterwards.
The recombinant plasmid pLTRB69regB was constructed as follows. The 1.3 kb DNA fragment was amplified from phage RB69 genome using two specific oligo primers: a direct primer, 5'-GGTTTAGTTGCTCAGGCGAGCA and a reverse primer, 5'-ATACGCATGGTGACCTTTCTTATC. The amplified DNA fragment was then digested with restriction endonucleases HincII and XbaI. The resulted 0.48 kb restriction fragment carrying the regB gene of phage RB69 was filled in, and the blunt-ended DNA fragment was inserted into the SacI site of plasmid vector pET21(+).
Plasmid pLPRB49regB was constructed as follows. A direct primer, 5'-CCGTTTGTATACTGAATTCGCAATTTG carrying EcoRI restriction site and a reverse primer, 5'-GCCCTATCTCGAGAAAGTCAAGC carrying XhoI restriction site were used to amplify a DNA fragment with gene regB of phage RB49. The amplified DNA fragment was then digested with restriction enzymes and the resulting 0.49 kb fragment was cloned into EcoRI–XhoI sites of plasmid vector pET21(+).
The nucleotide sequences of the cloned DNA fragments were confirmed by the dideoxy chain termination method (32). Standard procedures for the isolation and manipulation of plasmid DNA, and for the construction and identification of recombinant plasmids were used throughout (33). DNA restriction endonucleases, T4 DNA ligase and polymerase, DNA polymerase I (Klenow fragment), Taq DNA polymerase, Pfu DNA polymerase and CycleReaderTM DNA sequencing kit were obtained from Fermentas AB.
Assays of RegB endoribonuclease activity in phage-infected cells
To detect the RegB cleavage sites in the phage-induced transcripts, the E.coli BE cells were grown at 30°C to density 3 x 108 cells/ml in Luria–Bertani (LB) medium and then were infected with phages T4, RB69, RB49 or TuIa in the presence or in the absence of chloramphenicol. Chloramphenicol (150 μg/ml) was added 1 min prior to phage infection. The cells were collected 5 min after infection, lysed immediately and total cellular RNA was purified. Cleavages were analyzed by primer extension sequencing.
Assays of RegB endoribonuclease activity in plasmid–phage systems
The activities of RegB endoribonucleases of different phages were tested on transcripts induced either by phage infection or from recombinant plasmids containing phage genes. To test the activity of plasmid-encoded RegB endoribonucleases on phage-induced transcripts, the E.coli C41(DE3) cells harboring the recombinant plasmids pLPT4regB, pLTRB69regB or pLPRB49regB were grown in LB medium supplemented with ampicillin (40 μg/ml) at 30°C to A600 = 0.6. RegB was induced by the addition of isopropyl-?-D-thiogalactopyranoside (IPTG) to 1 mM, and 15 min later, the cells were infected either by phage RB49 or T4regBL52 (m.o.i. = 10). The cells were withdrawn at 4 or 5 min after infection, lysed immediately and total cellular RNA for primer extension sequencing was purified.
The activities of RegB endoribonucleases of different phages were also tested in reverse experiment. For this, E.coli C41(DE3) cells harboring recombinant plasmid pAZRB49vs.1' were grown in LB medium supplemented with ampicillin (40 μg/ml) at 30°C to A600 = 0.6. The RNA to be tested was induced from plasmid by the addition of IPTG to 1 mM, and 30 min later, the cells were infected by phages T4, RB69, TuIa, RB49 or RB42 (m.o.i. = 10). The cells were withdrawn at 5 min after infection, lysed immediately and total cellular RNA was purified. Cleavages were analyzed by primer extension sequencing.
RNase E cleavage assay
E.coli strains N3433 (rne+) and N3431 (rne–) were grown in LB medium supplemented with thymine (50 μg/ml) at 30°C to A600 = 0.5. With or without shifting of the cultures to 43°C for 30 min, cells were infected with T4D wild-type (m.o.i. = 10). The cells were withdrawn at 5 min after infection, lysed immediately and total cellular RNA was purified. RNase E cleavage was determined by primer extension sequencing.
RNA preparation and primer extension analysis of phage mRNAs
Total RNA from phage-infected E.coli cells was phenol extracted and used for the primer extension analysis or RNA sequencing under conditions of primer excess, using avian myeloblastosis virus reverse transcriptase, as described in (10,12). For details see Truncaite et al. (34). A total of eight synthetic oligonucleotides complementary to the coding sequences of genes of phages T4, RB69 (TuIa), RB49 were used to prime reverse transcriptase: Pr. T4regB(R1), 5'-GCGCGATCAAGAAGATGTTGAG, complementary to nucleotides 137–158 of the T4 regB gene; Pr. T4regB(R2), 5'-GCGGAGGCATACTCAGGAATTC, complementary to nucleotides 235–256 of the T4 regB gene; Pr. RB69regB(R1), 5'-GGCACGATCTAAGAGATGTTGAG, complementary to nucleotides 137–159 of the RB69 regB gene; Pr. RB49regB.1(R1), 5'-GCACATAATTCGACCTAGCGTACC, complementary to nucleotides 91–114 of the RB49 regB.1 gene; Pr. RB49regB(R1), 5'-GATCTTCGCATTGGTTTTGCGC, complementary to nucleotides 63–84 of the RB49 regB gene; Pr. RB49regB(R2), 5'-GCCCTATCACCAGAAAGTCAAGC, complementary to nucleotide 23–45 downstream of the termination codon of the RB49 regB gene; Pr. RB49vs.1(R1), 5'-GAATTGCTCGCCGTATTGATAGGC, complementary to nucleotides 136–159 of the RB49 vs.1 gene; Pr. RB49g43(R1), 5'-GTTGGCGTATATTCAACGTAACGCTC, complementary to nucleotides 76–101 of the RB49 gene 43.
RESULTS AND DISCUSSION
Sequences of gene regB of T4-related phages
The RegB endoribonuclease of bacteriophage T4 presents several unique aspects. It is the first description of the virus-encoded endoribonuclease that participates in the control of the expression of a number of phage early genes (10). This enzyme has almost absolute sequence specificity towards the GGAG tetranucleotides located in the SD regions of early mRNAs (14). Finally, RegB has a unique primary structure that presents no homology to other proteins, in particular to the other known RNases (35). The previous PCR analysis has shown the presence of regB homologues in the genomes of four T4-related phages, K3, RB70, T2 and T6 (20), but the nucleotide sequences have not been determined. In this work, we analyzed the conservation level of the gene regB in a wide group of T4 relatives. For this, we performed PCR and sequencing analysis of the genomic regions carrying gene regB of 36 T4-related phages.
A pair of primers based on the T4 gene regB sequence, Pr. 1–Pr. 2, and a pair of primers flanking the T4 gene regB, Pr. 3–Pr. 4, were used to amplify the analogues of gene regB of 36 T4-type phages. Using these primers, we obtained the PCR products of the expected sizes of 32 T4-related phages. The obtained PCR fragments were sequenced. The four phages, RB69, TuIa, RB42 and RB49, did not yield the PCR fragments with the primers used. A pair of primers based on the T4 gene vs.1 sequence yielded PCR fragment of RB69, which was sequenced and shown to contain gene vs.1 homologue. Additional primers were designed based on the newly obtained sequences of RB69 (see Materials and Methods). Using these primers, we were able to determine the sequence of gene regB of phages RB69 and TuIa. The sequence of gene vs.1 of pseudo T-even phage RB49 was available in GenBank (31). Based on this sequence, we designed primer and sequenced the upstream region of vs.1 of RB49. The RB49-specific primers were designed, and we defined the complete nucleotide sequence of gene regB of phage RB49. However, our attempts to obtain the sequence of gene regB of phage RB42 failed.
The sequences of the regB genes in 32 T4-related phages appeared to be very similar to each other. The deduced primary structures of RegB endoribonucleases of these phages were almost identical (98.6–100%) to the T4 RegB (Figure 1). Based on PCR analysis of generally conserved regions, most of these phages have been previously classified as T-evens (20). The phages RB69 and TuIa were also considered as the T-evens (26,28), but the DNA hybridization studies and sequence analysis of some of the genes indicated these phages could occupy an ‘intermediate’ position between T-even and pseudo T-even phages (26,36). The term ‘mezzo T-evens’ has been proposed to describe such a class of ‘intermediate’ T-even type phages (H. Krisch, personal communication). We also found that the RegB protein of RB69 and TuIa differed from the T4 RegB at a higher level that would be expected for T-evens: the primary structures of RegB endoribonucleases of phages TuIa and RB69 differed from the T4 RegB protein by 22.5%, but were identical to each other (Figure 1). The RegB of the pseudo T-even phage RB49 showed only 43.0% sequence identity with its T4 homologue and diverged mostly from the rest of the phages examined (Figure 1).
Figure 1. Amino acid sequence alignment of RegB endoribonucleases of T4-related phages. The RegB sequences of the phages were aligned with the T4 RegB sequence using the ClustalW program. A white background indicates amino acid motifs common to all of the T4-related subgroups. The sequences shown with black backgrounds are not well conserved. A dash indicates a space, which was inserted in the sequence to preserve the alignment. An asterisk (*) means that the residues in the column are identical; a double dot (:) indicates the conserved substitutions; a dot (.) indicates the semi-conserved substitutions.
It should be noted that we could not detect the regB gene in the genome of pseudo T-even phage RB42. The open reading frame (ORF) that we had sequenced upstream of the gene vs.1 of phage RB42 showed no homology to the T4 gene regB. Plasmid-induced transcripts carrying the GGAG motifs that were known to be well processed by T4 RegB were not cleaved after infection with RB42 (data not shown). This strongly suggested that the phage RB42 could lack the gene regB homologue. In support, it was recently shown that regB gene was also absent in the genome of phage KVP40, which is more distantly related to the T-evens (37). The maintenance of regB gene in most of the closely related T4-like phages might have been associated with a requirement for gpRegB in certain natural conditions that are not shared by phages distantly related to the T4.
Transcriptional and post-transcriptional control of regB expression in T4-related bacteriophages
Figure 2 shows the genes and the sites for transcriptional and post-transcriptional regulation in the regB region of four T4-type phages. In T4, the transcript for gene regB is initiated from a typical early promoter, PEregB, immediately upstream the gene (11). We analyzed the nucleotide sequence of the 5' flanking region of gene regB in 34 T4-related phages and found that all these phages possessed the promoter sequences immediately upstream the gene regB. The –35 and –10 promoter elements of PEregB of 32 T-even phages were identical to those of T4, and remarkably matched the T4 early promoter consensus elements –35 GTTTAC(a/ttt) and –10 TA(t/c)(a/t)AT spaced by 16–17 bp (2,38–41). The promoter sequences for gene regB of phages RB69 and TuIa also shared features of the T4 early promoters (Figure 3C). Thus, the regulation of regB transcription appeared to be similar in 34 phages tested.
Figure 2. Genes and promoters in the 1.10 kb genomic region with gene regB of bacteriophages T4, RB69 (TuIa) and RB49. The locations of early promoters are shown, as well as the positions of potential RegB cleavage sites within the SD sequences and in the coding sequences of regB. Vertical arrows show the sites susceptible to the RegB cleavage.
Figure 3. Primer extension analyses of transcripts for gene regB of phages T4, RB69 and TuIa. (A) Primer extension sequencing reaction of RNA isolated from E.coli BE cells at 5 min post-infection with bacteriophage T4 at 30°C. Primer extension reactions of RNA isolated at 1–15 min post-infection from the cells that were infected with phage T4 at 30°C are shown next to the sequencing reactions. (B) Primer extension sequencing reactions of RNA isolated from E.coli BE cells at 5 min post-infection with either phage RB69 or TuIa at 30°C. Primer extension sequencing reactions were performed using the primers Pr. T4regB(R1) (A) and Pr. RB69regB(R1) (B). The sequencing lanes are labeled with the dideoxynucleotides used in the sequencing reactions. The time (minutes) of post-infection that each RNA was isolated is noted at the top of the figures. The initiating nucleotides for the transcripts, the GGAG motifs within the SD sequences, the initiating codons, as well as the GGAG motifs in the coding region of gene regB mRNA are noted. (C) The nucleotide sequences of the 5' flanking region of gene regB of phages T4, RB69 and TuIa. The –35 and –10 regions of the early promoters, the initiating nucleotides for the transcripts, the GGAG motifs within the SD sequences, the initiation codons, as well as the GGAG motifs in coding regions of genes, are shown with black backgrounds. Vertical arrows denote the positions of RegB cleavage. Convergent arrows indicate inverted repeats. Asterisks indicate the termination codons for the upstream genes.
In RB49 genome, no T4 consensus sequences of either early or middle mode promoters were found (28,31). Analysis of the transcription of several regions of the RB49 genome, such as the 5' flanking regions of genes 43 and 32 (28,31), revealed the early promoters of RB49 to contain the sequences matching E.coli 70-dependent promoter sequence –35 cTTGACa and –10 TATAAT spaced by 16–18 bp (42–45). However, we could not detect any typical promoter sequence just upstream the gene regB, while the primer extension analyses of regB transcripts showed a weak transcription start site for this gene (Figures 4A and 5B). According to this site, we deduced promoter, PEregB, which contained the –35 sequence GTTTAAA and the –10 sequence TACTGG spaced by 15 bp (Figure 5). Thus, PEregB slightly matched the –35 element of T4 early promoters but had a poor –10 element, and the spacing region between them was too short in comparison to the T4 early promoters (2,38–41). Nevertheless, the in vitro transcription experiments demonstrated that the E.coli RNA polymerase could recognize this sequence on the PCR-generated DNA fragment and initiate transcription at the same position as in vivo (data not shown). Primer extension analysis of RB49 regB transcription showed that the PEregB acted as a very weak promoter (Figures 4A and 5B), and regB appeared to be transcribed mostly into the polycistronic transcripts from the more distal promoter PEregB.1 (Figure 2), which had a typical E.coli 70-dependent promoter sequence and was much more effective (Figure 5).
Figure 4. Primer extension analyses of phage RB49 transcript for gene regB. (A and B) Primer extension sequencing reactions of RNA isolated from E.coli BE cells at 5 min post-infection with RB49 at 30°C. Primer extension reactions of RNA isolated at 1–15 min post-infection from cells that were infected with RB49 at 30°C are shown next to the sequencing reactions. The sequencing lanes are labeled with dideoxynucleotides used in the sequencing reactions. The time (minutes) of post-infection that each RNA was isolated is noted at the top of the figures. (C) Primer extension sequencing of RNA isolated from E.coli N3433 (rne+) and N3431 (rne–) cells at 5 min post-infection with RB49 at 30 and 43°C. The GGAU motif within the SD sequence, the initiation codon for regB gene, the initiating nucleotide for the RegB-dependent transcript, as well as the 5' end nucleotide of RNase E processed transcript are noted. Primer extension sequencing reactions were performed using primers Pr. RB49regB(R1) (A) and Pr. RB49regB(R2) (B and C). (D) Nucleotide sequence flanking the RNase E cleavage site of the gene regB transcript and the potential secondary structures close to the cleavage site are shown. The arrow indicates the position of the RNase E cleavage. The 5' end nucleotide of RNase E processed transcript is shown with a black background. Asterisks indicate the termination codon for gene regB.
Figure 5. Primer extension sequencing of the transcripts for genes regB.1 (A), regB (B), vs.1 (C) and 43 (D) of phage RB49. Primer extension sequencing reactions of RNA isolated at 5 min post-infection from E.coli BE cells that were infected with RB49 in the absence or in the presence of chloramphenicol at 30°C. Sequencing of DNA fragment carrying gene 43 of phage RB49 is also presented (D). The initiating nucleotides for the transcripts, the potential RegB cleavage sites within the SD sequences, as well as initiation codons for the corresponding genes are noted. Convergent arrow indicates inverted repeat sequences. Primer extension reactions were performed using primers Pr. RB49regB.1(R1) (A), Pr. RB49regB(R1) (B), Pr. RB49vs.1(R1) (C) and Pr. RB49g43(R1) (D). The nucleotide sequences of the 5' flanking region of genes regB.1, regB, vs.1 and 43 of phage RB49 are shown at the bottom of the figure. The –35 and –10 regions of the early promoters, the initiating nucleotides for the transcripts, the GGAG and GGAU motifs within the SD sequences, as well as initiation codons are shown with black backgrounds. Vertical arrows denote the position of RegB cleavage. Convergent arrows indicate inverted repeats. Asterisks indicate the termination codons for the upstream genes.
A characteristic property of ribonucleases is the ability to autoregulate their synthesis in the cell. In E.coli, the genes for RNase III (46–48), RNase E (49–51), RNaseII (52,53) and PNPase (54–56) are known to be autoregulated or interregulated by controlling the degradation rate of their own mRNAs. Previous studies showed that the T4 endoribonuclease RegB efficiently cleaved its own mRNA within the GGAG motif located in the SD region and, less efficiently, the three additional GGAG motifs located in the coding sequence of the transcript for gene regB (11). Thirty-two T-evens analyzed in this study also carried the GGAG motif in the SD region and contained three or at least two GGAG motifs within the proximal part of the coding sequence of gene regB. Phages RB69 and TuIa contained the GGAG motif in the SD region and only one GGAG downstream from the initiation codon AUG (Figure 2). The SD region of RB49 gene regB contained a motif GGAU. In T4, only two exceptions were found where the RegB cleaved a GGAU sequence (13). The coding sequence of RB49 regB mRNA lacked the putative RegB cleavage sites at the positions observed for T4, but had two GGAU motifs and the GGAG motif within the distal part of the coding sequence of regB transcript (Figure 2). Thus, autoregulation of expression seemed to be natural to the phages studied, although the concentration of the putative RegB target sites within the regB transcripts was different.
We tested the RegB nucleases of phages RB69, TuIa and RB49, that exhibited higher degree of divergence from T4, for their activity towards GGAG and GGAU motifs located in the SD regions and in the coding sequences of their regB transcripts. Figure 3A and B shows the results of primer extension sequencing on RNAs extracted from the cells after infection with T4, RB69 and TuIa. Two 5' truncated RNA termini were detected in every case. The first cut occurred within the SD sequence and the second one laid in the coding sequence downstream from AUG codon in the cases of RB69 and TuIa, as well as in T4.
Figure 4A shows the primer extension sequencing and the kinetics of accumulation of RB49 regB mRNAs. The 5' end of mRNA assigned to the transcription initiation appeared as soon as the first minute of infection, while the 5' end of mRNA cleaved at the middle of the GGAU motif of the SD region was detected at the second minute of infection. Primer extension sequencing of the RB49 mRNAs for gene regB extracted from chloramphenicol-treated cells indicated that the cleavage was phage induced (Figure 5B). Thus, we can conclude that autoregulation of the expression at the post-transcriptional level is common to the regB of phages RB69, TuIa and RB49.
Two GGAU motifs as well as the GGAG located in the coding sequence of RB49 regB were resistant to RB49 RegB nucleolytic activity (data not shown). While seeking for the RegB-dependent cleavages in the coding sequence of the transcript for RB49 gene regB, the additional nucleolytic cut was observed within the distal part of the mRNA (Figure 4B). Primer extension analysis showed that the 5' end of the truncated RB49 regB mRNA appeared at 3 min of infection and continued to accumulate onward. The 5' end of truncated RNA was seen even when RB49 infection was carried out in the presence of chloramphenicol (data not shown) indicating that this endonucleolytic cleavage was caused by a host-encoded endoribonuclease. The sequence where the cut occurred matched the processing site for the E.coli RNase E (3,23,57–60): the region was A–U rich and three stable stem–loops were supposed adjacent (Figure 4D).
To test whether the truncated mRNA could be the product of E.coli RNase E cleavage, total RNA was extracted from isogenic E.coli wild-type and rne (ts) strains after infection with phage RB49. The extracted mRNA was analyzed by primer extension sequencing. In the wild-type background, the truncated mRNA of RB49 regB was detected either in 30°C or in 43°C. In the rne (ts) background at 43°C, the 5' ends of truncated mRNA were not observed, indicating that the 5' ends were generated by RNase E-dependent cleavage and not by any other endonucleolytic event (Figure 4C). Therefore, we can conclude that the RegB of RB49 controls its biosynthesis by attacking its own mRNA within GGAU motif in the SD region, but in addition, the E.coli ribonuclease E is utilized for the degradation of regB transcripts of RB49. We looked for the RNase E processing sites in the regB transcripts of phages T4 and RB69, but the systematic primer extension analysis did not reveal RNase E-dependent cleavages.
E.coli RNase E was originally implicated in processing of T4 transcripts for genes 32 (57,58) and -gt (23). Furthermore, this ribonuclease was shown to have a major role in the degradation of many T4 messages (9). The RNase E-dependent processing sites in the gene 32-transcription unit was shown to be conserved in six T4-related phages (23,61). In spite of the sequence heterogeneity in this region, RNase E cleaved mRNAs for gene 32 of these phages (23). However, in case of phage RB49 infection, unlike that of T4, the host RNase E was shown to have no major role in processing the gene 32-transcription unit (31). Therefore, we present perhaps the first clear evidence of the E.coli RNase E activity towards phage RB49 mRNAs.
Specificities of different RegB endoribonucleases
The RegB nuclease of bacteriophage T4 shows high specificity to the GGAG sequence but exhibits different activity in vivo towards different classes of mRNA (14). The GGAG-carrying RNAs that are uncut during T4 infection are also resistant to RegB in the two-plasmid systems in vitro indicating that the information identifying the substrate for RegB is carried in cis by RNA molecule (14,17,62). However, clear sequence motif outside GGAG distinguishing substrates and non-substrates of RegB has not been identified. It was shown that RegB preferentially cleaves GGAG motifs involved in a particular secondary structure, the formation of which is favored by the ribosomal protein S1 (17,63).
However, little information is available in the literature concerning the protein determinants of RegB that contribute to the recognition specificity and cleavage activity of this enzyme. Examination of the T4 regB mutants constructed by a site-directed mutagenesis revealed the potent catalytic residues His-48 and His-68 (35,64). The Arg-52 was proposed to be important for the binding of the substrate RNA. The comparison of the primary structures of 35 RegB ribonucleases determined in this study showed that all these three residues were conserved between the 35 T4-related phages tested, except for RB49 RegB, where histidine H68 was changed to asparagine (Figure 1). Moreover, the RegB of the more distant T4 relatives had multiple variations in primary structure of the protein, and the sequence of RB49 RegB was shorter than the RegB of T4 by 13 amino acids. Therefore, we wondered if the RegB endoribonucleases encoded by distant T4-like phages retained the same functional properties as the T4 RegB.
As it was already mentioned, we found that the RegB endonucleases of RB69 and TuIa cleaved the GGAG motifs within the SD regions and in the coding sequences of their mRNAs for gene regB. The RegB of RB49 processed only the GGAU motif located within the ribosome-binding region of its regB mRNA but not the GGAU or GGAG motifs located within the coding region. In order to test the ability of RB49 RegB to cleave the GGAG motifs, we performed primer extension sequencing of RB49 transcripts for genes regB.1 and vs.1 (Figure 2), as well as for gene 43 (31), that were known to carry GGAG motifs in their SD regions. The mRNA for gene regB.1 was processed within SD sequence by ribonuclease RegB of RB49 (Figure 5A), but the mRNAs of genes vs.1 and 43 were resistant (Figure 5C and D). To ensure that the observed cuts in the SD sequences of genes regB.1 and regB were caused by endoribonuclease RegB and not by E.coli endonucleolytic activity, the primer extension sequencing experiments were also carried on RNAs extracted from the cells after infection with RB49 in the presence of chloramphenicol. The 5' end of truncated mRNA did not appear in both cases (Figure 5A and B) indicating that the activity was phage induced.
The noticeable selectivity against the GGAG and GGAU motifs observed for RB49 RegB might depend on the differences in the primary structure of this enzyme. To test this, we examined the capability of RegB of phages T4, RB69 and TuIa to cleave those GGAG-carrying mRNAs that were not cleaved by RB49 RegB. For this, we cloned the proximal part of gene vs.1 of phage RB49 into an inducible plasmid pET21(+) and transformed E.coli C41(DE3) cells with the resulted plasmid pAZRB49vs.1'. The transcripts for the proximal part of RB49 gene vs.1 were induced by addition of IPTG to the medium, and 15 min later, the cells were infected with one of the four phages. Total RNA was extracted, and the RegB cleavage sites were mapped by RNA sequencing of plasmid-induced transcripts. During T4, RB69 or TuIa infection, the RB49 vs.1 transcript induced from plasmid was cleaved in the SD region, though the efficiency of T4 RegB cleavage was much lower than those of RB69 and TuIa (Figure 6A). As expected, the plasmid-induced RB49 vs.1 transcript stayed resistant during infection of RB49. The RegB-dependent processing event was also not observed after infection with phage RB42 (Figure 6A).
Figure 6. Susceptibility of RB49 transcripts for genes vs.1 and 43 to the RegB endoribonucleases of different T4-related bacteriophages. (A) Primer extension sequencing of RNA isolated from E.coli C41(DE3) cells, harboring recombinant plasmid pAZRB49vs.1', after 5 min post-infection at 30°C with one of the five phages indicated above the figure. The transcript for the gene vs.1 was induced from the plasmid by the addition of IPTG 30 min before infection. The control experiment without phage infection is also shown. Cleavages were detected by primer extension sequencing using T7 terminator primer 5'-GCTAGTTATTGCTCAGCG. (B and C) Primer extension sequencing of RNA isolated from E.coli C41(DE3) cells, harboring one of the three recombinant plasmids indicated above the figures, at 5 min post-infection with phage RB49 at 30°C. RegB endoribonuclease of phages T4, RB69 or RB49 was induced from the corresponding plasmids by the addition of IPTG 15 min before infection. The control experiment shows the primer extension sequencing of RNA isolated from E.coli C41(DE3) cells at 5 min post-infection with RB49 at 30°C. The sequencing lanes are labeled with dideoxynucleotides used in the sequencing reactions. The initiating nucleotides for the RB49 transcripts of genes vs.1 (B) and 43 (C), the GGAG motifs within the SD sequences, the initiation codons for the corresponding genes are noted. Primer extension sequencing reactions were performed using primers Pr. RB49vs.1(R1) (B) and Pr. RB49g43(R1) (C).
In the reverse experiment, we cloned the regB genes of phages T4, RB69 or RB49 into the inducible plasmid pET21(+). The RegB endoribonuclease was induced by addition of IPTG into the medium with E.coli cells carrying one of the recombinant plasmids with a cloned regB gene, and later the cells were infected with RB49. The total RNA was extracted and used for primer extension sequencing of gene vs.1 and gene 43 mRNAs of RB49. The plasmid-induced RegB endoribonucleases of T4 and RB69 cleaved the transcripts for gene vs.1 and gene 43 of RB49 at the GGAG motif present in their SD regions, while the RegB of RB49 had no effect on these mRNAs again (Figure 6B and C).
To test whether the RB49 RegB induced from the recombinant plasmid could process GGAG motifs located in the coding sequence of the T4 transcripts for gene regB, we used the E.coli C41(DE3) cells carrying the plasmid pLPRB49regB. For the control experiments, we also used E.coli C41(DE3) carrying the plasmid pET21(+) and E.coli C41(DE3) carrying the plasmids pLPT4regB or pLTRB69regB. The RegB endoribonucleases from the recombinant plasmids were induced by addition of IPTG. Later, the cells were infected with either bacteriophage T4 wild-type or the nuclease-deficient phage T4 regBL52. Total mRNA was extracted in each case of infection, and the cleavage sites were detected by primer extension sequencing. The results showed that the RB49 RegB could process the same motifs of the T4 regB transcripts as the T4 RegB but the efficiency of processing differed. The first and the third T4 RegB processing sites located in the coding sequence of regB transcript were more susceptible to the T4 or RB69 RegB ribonucleolytic attack in comparison to the second one (Figure 7A, C and D). Whereas, the RB49 RegB cleaved the second GGAG motif more efficiently than the SD, the first and the third ones (Figure 7E).
Figure 7. Susceptibility of the T4 transcripts for gene regB to the RegB endoribonuclease of phages T4, RB69 and RB49. Primer extension sequencing of mRNA for gene regB isolated from the E.coli C41(DE3) cells harboring plasmid pET21(+) at 4 min post-infection by T4 wild-type phage at 30°C (A). Primer extension sequencing of the T4regBL52 mRNA for gene regB isolated at 4 min post-infection by phage at 30°C from the E.coli C41(DE3) cells harboring plasmid pET21(+) (B) or the recombinant plasmids pLPT4regB (C), pLTRB69regB (D) and pLPRB49regB (E). Primer Pr. T4regB(R2) was used in the sequencing reactions. The sequencing lanes are labeled with the dideoxynucleotides used in the sequencing reactions. The initiating nucleotides for the transcripts, the GGAG motifs within the SD sequences, as well as the GGAG motifs in the coding region of gene regB mRNA are noted. (F) The nucleotide sequence of the 5' part of mRNA for gene regB of bacteriophage T4. The GGAG motif within the SD sequence, the initiation codon, as well as the GGAG motifs in the coding region are shown with black backgrounds. Vertical arrows denote the positions of RegB cleavage.
Taken together, the results of these experiments clearly indicate that four homologous RegB endoribonucleases encoded by phages T4, RB69, TuIa and RB49 possess different capability to recognize GGAG/U motifs flanked by the same sequences. Some GGAG-carrying transcripts that are not cleaved by the RB49 RegB can be recognized and processed by the RegB of phages T4, RB69 or TuIa though with different efficiency. Different susceptibility of the same mRNAs to the RegB endoribonucleases of more distant T4 relatives could clearly be related to the differences in the primary structure of these proteins. It seems possible to propose that the highly diverged RegB of RB49 has more tight sequence requirements for the processing site. We expect the comparative studies of the structure–function relationships between the RegB homologues may help to detect the specificity determinants carried by the RegB protein.
ACKNOWLEDGEMENTS
We thank Dr Lindsay W. Black, Dr Philippe Régnier and Dr Kenneth N. Kreuzer for E.coli strains, Dr Karin Carlson, Dr William B. Wood and Dr Marc Uzan for phages. This work was supported in part by the Lithuanian State Science and Studies Foundation.
REFERENCES
Ueno,H. and Yonesaki,T. ( (2001) ) Recognition and specific degradation of bacteriophage T4 mRNAs. Genetics, , 158, , 7–17.
Miller,E.S., Kutter,E., Mosig,G., Arisaka,F., Kunisawa,T. and Rüger,W. ( (2003) ) Bacteriophage T4 genome. Microbiol. Mol. Biol. Rev., , 67, , 86–156.
Otsuka,Y., Ueno,H. and Yonesaki,T. ( (2003) ) Escherichia coli endoribonucleases involved in cleavage of bacteriophage T4 mRNAs. J. Bacteriol., , 185, , 983–990.
Deutscher,M.P. and Li,Z. ( (2001) ) Exoribonucleases and their multiple roles in RNA metabolism. Prog. Nucleic Acid Res. Mol. Biol., , 66, , 67–105.
Zuo,Y. and Deutscher,M.P. ( (2001) ) Exoribonuclease superfamilies: structural analysis and phylogenetic distribution. Nucleic Acids Res., , 29, , 1017–1026.
Deutscher,M.P. ( (1993) ) Ribonuclease multiplicity, diversity and complexity. J. Biol. Chem., , 268, , 13011–13014.
Kushner,S.R. ( (2002) ) mRNA decay in Escherichia coli comes of age. J. Mol. Biol., , 184, , 4658–4665.
Kennel,D. ( (2002) ) Processing endoribonucleases and mRNA degradation in bacteria. J. Bacteriol., , 184, , 4645–4657.
Mudd,E.A., Carpousis,A.J. and Krisch,H.M. ( (1990) ) E.coli RNase E has a role in the decay of bacteriophage T4 mRNA. Genes Dev., , 4, , 873–881.
Uzan,M., Favre,R. and Brody,E. ( (1988) ) A nuclease that cuts specifically in the ribosome binding site of some T4 mRNAs. Proc. Natl Acad. Sci. USA, , 85, , 8895–8899.
Ruckman,J., Parma,D., Tuerk,C., Hall,D.H. and Gold,L. ( (1989) ) Identification of a T4 gene required for bacteriophage mRNA processing. New Biol., , 1, , 54–65.
Sanson,B. and Uzan,M. ( (1993) ) Dual role of the sequence-specific bacteriophage T4 endoribonuclease RegB: mRNA inactivation and mRNA destabilization. J. Mol. Biol., , 233, , 429–446.
Sanson,B. and Uzan,M. ( (1995) ) Post-transcriptional controls in bacteriophage T4: roles of the sequence-specific endoribonuclease RegB. FEMS Microbiol. Rev., , 17, , 141–150.
Sanson,B., Hu,R.-M., Troitskaya,E., Mathy,N. and Uzan,M. ( (2000) ) Endoribonuclease RegB from bacteriophage T4 is necessary for the degradation of early but not middle or late mRNAs. J. Mol. Biol., , 297, , 1063–1074.
Truncaite,L. ( (2001) ) Deletional and transcriptional analysis of the genomic region between genes 30 and 31 of bacteriophage T4. PhD Thesis, Vilnius, Lithuania.
Ruckman,J., Ringquist,S., Brody,E. and Gold,L. ( (1994) ) The bacteriophage T4 regB ribonuclease. Stimulation of the purified enzyme by ribosomal protein S1. J. Biol. Chem., , 269, , 26655–26662.
Lebars,I., Hu,R.M., Lallemand,J.Y., Uzan,M. and Bontems,F. ( (2001) ) Role of the substrate conformation and of the S1 protein in the cleavage efficiency of the T4 endoribonuclease RegB. J. Biol. Chem., , 276, , 13264–13272.
Chace,K.V. and Hall,D.H. ( (1975) ) Characterization of new regulatory mutants of bacteriophage T4. II. New class of mutants. J. Virol., , 15, , 929–945.
Valerie,K., Stevens,J., Lynch,M., Henderson,E.E. and de Riel,J.K. ( (1986) ) Nucleotide sequence and analysis of the 58.3 to 65.5-kb early region of bacteriophage T4. Nucleic Acids Res., , 14, , 8637–8654.
Repoila,F., Tétart,F., Bouet,J.Y. and Krisch,H.M. ( (1994) ) Genomic polymorphism in the T-even bacteriophages. EMBO J., , 13, , 4181–4192.
Kutter,E., Gachechiladze,K., Poglazov,A., Marusich,E., Shneider,M., Aronsson,P., Napuli,A., Porter,D. and Mesyanzhinov,V. ( (1996) ) Evolution of T4-related phages. Virus Genes, , 11, , 285–297.
Ackermann,A.W. and Krisch,H.M. ( (1997) ) A catalogue of T4-type bacteriophages. Arch. Virol., , 142, , 2329–2345.
Loayza,D., Carpousis,A.J. and Krisch,H.M. ( (1991) ) Gene 32 transcription and mRNA processing in T4-related bacteriophages. Mol. Microbiol., , 5, , 715–725.
Selick,H.E., Stormo,G.D., Dyson,R.L. and Alberts,B.M. ( (1993) ) Analysis of five presumptive protein-coding sequences clustered between the primosome genes, 41 and 61, of bacteriophages T4, T2, and T6. J. Virol., , 67, , 2305–2316.
Monod,C., Repoila,F., Kutateladze,M., Tétart,F. and Krisch,H.M. ( (1997) ) The genome of the pseudo T-even bacteriophages, a diverse group that resembles T4. J. Mol. Biol., , 267, , 237–249.
Tétart,F., Desplats,C., Kutateladze,M., Monod,C., Ackermann,H.W. and Krisch,H.M. ( (2001) ) Phylogeny of the major head and tail genes of the wide-ranging T4-type bacteriophages. J. Bacteriol., , 183, , 358–366.
Hambly,E., Tétart,F., Desplats,C., Wilson,W.H., Krisch,H.M. and Mann,N.H. ( (2001) ) A conserved genetic module that encodes the major virion components in both the coliphage T4 and the marine cyanophage S-PM2. Proc. Natl Acad. Sci. USA, , 98, , 11411–11416.
Desplats,C. and Krisch,H.M. ( (2003) ) The diversity and evolution of the T4-type bacteriophages. Res. Microbiol., , 154, , 259–267.
Miroux,B. and Walker,J.E. ( (1996) ) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol., , 260, , 289–298.
Kricker,M. ( (1994) ) Sequencing of genomic T4 DNA. In Karam,J.D. (editor-in-chief), Molecular Biology of Bacteriophage T4. ASM Press, Washington, DC, pp. 463–464.
Desplats,C., Dez,C., Tétart,F., Eleaume,H. and Krisch,H.M. ( (2002) ) Snapshot of the genome of the pseudo-T-even bacteriophage RB49. J. Bacteriol., , 184, , 2789–2804.
Sanger,F., Nicklen,S. and Coulson,A.R. ( (1977) ) DNA sequencing with chain-terminating inhibitors. Proc. Natl Acad. Sci. USA, , 74, , 5463–5467.
Sambrook,J., Fritsch,E.F. and Maniatis,T. ( (1989) ) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Truncaite,L., Pieiniene,L., Kolesinskiene,G., Zajankauskaite,A., Driukas,A., Klausa,V. and Nivinskas,R. ( (2003) ) Twelve new MotA-dependent middle promoters of bacteriophage T4: consensus sequence revised. J. Mol. Biol., , 327, , 335–346.
Sa?da,F., Uzan,M. and Bontems,F. ( (2003) ) The phage T4 restriction endoribonuclease RegB: a cyclizing enzyme that requires two histidines to be fully active. Nucleic Acids Res., , 31, , 2751–2758.
Yeh,L.S., Hsu,T. and Karam,J.D. ( (1998) ) Divergence of a DNA replication gene cluster in the T4-related bacteriophage RB69. J. Bacteriol., , 180, , 2005–2013.
Miller,E.S., Heidelberg,J.F., Eisen,J.A., Nelson,W.C., Durkin,A.S., Ciecko,A., Feldblyum,T.V., White,O., Paulsen,I.T., Nierman,W.C., Lee,J., Szczypinski,B. and Fraser,C.M. ( (2003) ) Complete genome sequence of the broad-host-range vibriophage KVP40: comparative genomics of a T4-related bacteriophage. J. Bacteriol., , 185, , 5220–5233.
Liebig,H.D. and Rüger,W. ( (1989) ) Bacteriophage T4 early promoter regions. Consensus sequences of promoters and ribosome-binding sites. J. Mol. Biol., , 208, , 517–536.
Wilkens,K. and Rüger,W. ( (1994) ) Transcription from early promoters. In Karam,J.D. (editor-in-chief), Molecular Biology of Bacteriophage T4. ASM Press, Washington, DC, pp. 132–141.
Wilkens,K. and Rüger,W. ( (1996) ) Characterization of bacteriophage T4 early promoters in vivo with a new promoter probe vector. Plasmid, , 35, , 108–120.
Sommer,N., Salniene,V., Gineikiene,E., Nivinskas,R. and Rüger,W. ( (2000) ) T4 early promoter strength probed in vivo with unribosylated and ADP-ribosylated Escherichia coli RNA polymerase: a mutation analysis. Microbiology, , 146, , 2643–2653.
Rosenberg,M. and Court,D. ( (1979) ) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu. Rev. Genet., , 13, , 319–353.
Hawley,D.K. and McClure,W.R. ( (1983) ) Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res., , 11, , 2237–2255.
Harley,C.B. and Reynolds,R.P. ( (1987) ) Analysis of E.coli promoter sequences. Nucleic Acids Res., , 15, , 2343–2361.
Lisser,S. and Margalit,H. ( (1993) ) Compilation of E.coli mRNA promoter sequences. Nucleic Acids Res., , 21, , 1507–1516.
Bardwell,J.C.A., Régnier,P., Chen,S.-M., Nakamura,Y., Grunberg-Manago,M. and Court,D.L. ( (1989) ) Autoregulation of RNase III operon by mRNA processing. EMBO J., , 8, , 3401–3407.
Matsunaga,J., Dyer,M., Simons,E.L. and Simons,R.W. ( (1996) ) Expression and regulation of the rnc and pdxJ operons of Escherichia coli. Mol. Microbiol., , 22, , 977–989.
Matsunaga,J., Simons,E.L. and Simons,R.W. ( (1997) ) Escherichia coli RNase III (rnc) autoregulation occurs independently of rnc gene translation. Mol. Microbiol., , 26, , 1125–1135.
Jain,C. and Belasco,J.G. ( (1995) ) RNase E autoregulates its synthesis by controlling the degradation rate of its own mRNA in Escherichia coli: unusual sensitivity of the rne transcript to RNase E activity. Genes Dev., , 9, , 84–96.
Diwa,A., Bricker,A.L., Jain,C. and Belasco,J.G. ( (2000) ) An evolutionarily conserved RNA stem–loop functions as a sensor that directs feedback regulation of RNase E gene expression. Genes Dev., , 14, , 1249–1260.
Ow,M.C., Liu,Q., Mohanty,B.K., Andrew,M.E., Maples,V.F. and Kushner,S.R. ( (2002) ) RNase E levels in Escherichia coli are controlled by a complex regulatory system that involves transcription of the rne gene from three promoters. Mol. Microbiol., , 43, , 159–171.
Zilh?o,R., Régnier,P. and Arraiano,C.M. ( (1995) ) The role of endonucleases in the expression of ribonuclease II in Escherichia coli. FEMS Microbiol. Lett., , 130, , 237–244.
Zilh?o,R., Cairr?o,F., Régnier,P. and Arraiano,C.M. ( (1996) ) PNPase modulates RNase II expression in Escherichia coli: implications for mRNA decay and cell metabolism. Mol. Microbiol., , 20, , 1033–1042.
Robert-LeMeur,M. and Portier,C. ( (1992) ) E.coli polynucleotide phosphorylase expression is autoregulated through an RNase III-dependent mechanism. EMBO J., , 11, , 2633–2641.
Robert-LeMeur,M. and Portier,C. ( (1994) ) Polynucleotide phosphorylase of Escherichia coli induces the degradation of its RNase III processed messenger by preventing its translation. Nucleic Acids Res., , 22, , 397–403.
Jarrige,A.C., Mathy,N. and Portier,C. ( (2001) ) PNPase autocontrols its expression by degrading a double-stranded structure in the pnp mRNA leader. EMBO J., , 20, , 6845–6855.
Mudd,E.A., Prentki,P., Belin,D. and Krisch,H.M. ( (1988) ) Processing of unstable bacteriophage T4 gene 32 mRNAs into a stable species requires Escherichia coli ribonuclease E. EMBO J., , 7, , 3601–3607.
Carpousis,A.J., Mudd,E.A. and Krisch,H.M. ( (1989) ) Transcription and messenger RNA processing upstream of bacteriophage T4 gene 32. Mol. Gen. Genet., , 219, , 39–48.
Ehretsmann,C.P., Carpousis,A.J. and Krisch,H.M. ( (1992) ) Specificity of Escherichia coli endoribonuclease RNase E: in vivo and in vitro analysis of mutants in a bacteriophage T4 mRNA processing site. Genes Dev., , 6, , 149–159.
McDowall,K.J., Lin-Chao,S. and Cohen,S.N. ( (1994) ) A + U content rather than a particular nucleotide order determines the specificity of RNase E cleavage. J. Biol. Chem., , 269, , 10790–10796.
Carpousis,A.J. and Krisch,H.M. ( (1994) ) mRNA processing and degradation. In Karam,J.D. (editor-in-chief), Molecular Biology of Bacteriophage T4. ASM Press, Washington, DC, pp. 176–181.
Jayasena,V.K., Brown,D., Shtatland,T. and Gold,L. ( (1996) ) In vitro selection of RNA specifically cleaved by bacteriophage T4 RegB endonuclease. Biochemistry, , 35, , 2349–2356.
Bisaglia,M., Laalami,S., Uzan,M. and Bontems,F. ( (2003) ) Activation of the RegB endoribonuclease by the S1 ribosomal protein is due to cooperation between the S1 four C-terminal modules in a substrate-dependent manner. J. Biol. Chem., , 278, , 15261–15271.
Sa?da,F., Odaert,B., Uzan,M. and Bontems,F. ( (2004) ) First structural investigation of the restriction ribonuclease RegB: NMR spectroscopic conditions, 13C/15N double-isotopic labelling and two-dimensional heteronuclear spectra. Protein Expr. Purif., , 34, , 158–165.(Lina Pieiniene, Lidija Truncaite, Aureli)