Evidence that the Promoter Can Influence Assembly of Antitermination Complexes at Downstream RNA Sites
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《细菌学杂志》
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109,Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242
ABSTRACT
The N protein of phage acts with Escherichia coli Nus proteins at RNA sites, NUT, to modify RNA polymerase (RNAP) to a form that overrides transcription terminators. These interactions have been thought to be the primary determinants of the effectiveness of N-mediated antitermination. We present evidence that the associated promoter, in this case the early PR promoter, can influence N-mediated modification of RNAP even though modification occurs at a site (NUTR) located downstream of the intervening cro gene. As predicted by genetic analysis and confirmed by in vivo transcription studies, a combination of two mutations in PR, at positions –14 and –45 (yielding PR-GA), reduces effectiveness of N modification, while an additional mutation at position –30 (yielding PR-GCA) suppresses this effect. In vivo, the level of PR-GA-directed transcription was twice as great as the wild-type level, while transcription directed by PR-GCA was the same as that directed by the wild-type promoter. However, the rate of open complex formation at PR-GA in vitro was roughly one-third the rate for wild-type PR. We ascribe this apparent discrepancy to an effect of the mutations in PR-GCA on promoter clearance. Based on the in vivo experiments, one plausible explanation for our results is that increased transcription can lead to a failure to form active antitermination complexes with NUT RNA, which, in turn, causes failure to read through downstream termination sites. By blocking antitermination and thus expression of late functions, the effect of increased transcription through nut sites could be physiologically important in maintaining proper regulation of gene expression early in phage development.
INTRODUCTION
Expression of the delayed-early genes of coliphage is regulated by transcription termination and antitermination (19). Transcription initiating at the two early promoters PL and PR partially terminates at terminators tL1 and tR1, respectively (Fig. 1A). Early transcription from PL results in production of N protein, which acts together with a number of host factors (Nus proteins) at NUT sites in nascent-early transcripts with RNA polymerase (RNAP) to form a transcription complex that is resistant to both intrinsic (Rho-independent) and Rho-dependent terminators. Escherichia coli proteins that combine with N and NUT RNA to modify RNAP include NusA, NusB, ribosomal protein S10 (NusE), and NusG (10, 23, 40, 43, 55).
Two classes of mutations in nus genes have been isolated. Class 1 mutations reduce effective N action, while class 2 mutations suppress the action of class 1 mutations by restoring the effectiveness of N in the presence of a class 1 mutation. For example, the failure of E. coli nusA1 or nusE71 mutants to support N action can be suppressed by class 2 mutations in either nusB or nusG (50, 52). In addition to these class 2 nus mutations, a point mutation in rpoA, encoding the subunit of RNAP, suppresses the effects of nusA1 or nusE71 (47).
With the exception of N, all phage-encoded factors required for lytic development of ultimately depend on transcription from PR (21), which is required for expression of cro, cII, replication genes O and P, and the Q gene. Q protein, in turn, is required for the expression of late genes, including those responsible for lysis and morphogenesis (44). The Q protein, like N, is an antitermination protein. However, Q acts through a DNA site, qut, rather than an RNA site, to modify transcription complexes initiating at the late PR' promoter (Fig. 1).
In the absence of N, about 40% of the transcripts initiating at PR transcend tR1 and terminate within the nin terminator region (Fig. 1A) (5, 8). Two mutations that permit to form plaques on nus mutants at high temperature reduce or eliminate the requirement for N-mediated antitermination. These are nin5, a deletion of the nin region, (8) and Pbyp, a mutation generating a new promoter (6) that directs constitutive expression of Q (3, 29). Mutations in the nutR region [e.g., boxA(Con)] and in N (e.g., NpunA1,133) also suppress the effects of nus mutations on phage production (24, 46).
The nut sites are proficient at promoting N-mediated antitermination when placed downstream of promoters other than those of , such as the E. coli gal promoter (12). Thus, in the case of N-mediated antitermination, all of the interactions influencing modification of RNAP to an antiterminating form have been thought to occur at the NUT sites. (Sequences in the RNA are indicated by having every letter capitalized.) In this communication, study of a mutant PR promoter altered by two base changes shows that events occurring at the promoter influence the effectiveness of N modification. Data presented here address two ways that these mutations in PR influence gene expression. First, we demonstrate in vivo that increased transcription observed with the mutant promoter results in failure to modify RNAP at NUTR, which in turn prevents antitermination at downstream terminators in the nin region. Consequently, Q gene expression is not sufficient to allow transcription of late genes. Second, we investigate in vitro how transcription increases in spite of the fact that these mutations in PR decrease the efficiency of open complex formation.
MATERIALS AND METHODS
Bacterial strains and phages. Relevant genotypes and derivations of the E. coli strains and derivatives used in this study are listed in Table 1. Plasmid pJGN was constructed by inserting the N gene in place of the Q gene in pJG100 (31), a plasmid that also has the lacIq gene.
Media and reagents. For most experiments, bacteria were grown in L (Luria) broth. In phage burst experiments, L broth was supplemented with 0.2% maltose. For -galactosidase assays, cells were grown in M9 medium supplemented with 0.2% fructose and 0.1% Casamino Acids. The minimal medium in the phage burst experiments was M9 supplemented with 0.2% maltose. Antibiotics were used at the following concentrations: 50 μg/ml for ampicillin and 30 μg/ml for kanamycin.
The following reagents were purchased from the indicated companies: oligonucleotides (Invitrogen), RNAP holoenzyme (Epicenter), restriction endonucleases (New England BioLabs), Taq polymerase, and RNase free DNase I (Roche Biochemicals).
Phage construction. derivatives with mutations in the PR region were constructed using either of two methods to recombine the DNA with the designed changes into prophages. Both methods exploit the recombination functions of phage to obtain enhanced homologous recombination.
Method A. The procedure developed by Murphy (39) was used to cross designed mutations into the cI857x13 prophage (13) of strain K9227. In this strain, recombination genes are substituted for the recBCD locus and are controlled by the lac promoter.
Method B. The procedure of Court et al. (9) was used to cross designed mutations contained within single-stranded oligonucleotides into the cI857x13 prophage in strain K120. The cI857x13 prophage supplied recombination functions. PR-GA (containing two mutations in PR, at positions –14 and –45) derivatives containing additional mutations, including boxA(Con), byp, and NpunA1,133, were constructed using the method outlined by Oppenheim et al. (41) to cross sequences from single-stranded DNA to the phage genome. PR-GAnin was constructed by recombination between PR-GA and imm434nin with standard phage genetic techniques.
Single-burst and EOP experiments. Production of phage after infection was assayed with single-step growth experiments (17). Efficiency of plating (EOP) was determined by dividing the plaque count on a lawn of the mutant strain by that on a lawn of K37, wild type for nusA, nusG, and rpoD (for details, see reference 22).
Reporter fusion construction. The reporter fusions were constructed by replacing the wild-type PR in a reporter construct with the PR promoter variants. The reporter construct has the cI-PR-cro region of followed by cat-sacB fused to the chromosomal lacZ gene and an adjacent kanR marker (51). The recombineering system of Court et al. (9) was used to generate cro-lacZ fusions under the control of each of the PR derivatives. The constructs were then transduced into nusA+ and nusA1 strains for analysis by use of KanR for selection.
-Galactosidase assays. Bacteria were grown overnight at 32°C in M9 medium supplemented with 0.2% fructose and 0.1% Casamino Acids. Overnight cultures were diluted into the same medium, grown at 32°C to early log phase, and then grown for 1 h at 40°C. Bacteria were harvested, and -galactosidase activity was assayed according to the Miller protocol (37).
Quantitative real-time RT-PCR. Bacteria were grown to mid-log phase in L broth supplemented with 0.2% maltose, concentrated by centrifugation, resuspended in a reduced volume, and incubated with various derivatives on ice at a multiplicity of infection of 5 to allow adsorption. After 20 min, infected bacteria were diluted in L broth supplemented with 10 mM MgSO4 and grown at 32°C for 30 min. Total RNA was prepared from the infected bacteria by use of the TRIzol procedure (Invitrogen). After DNase I digestion, the RNA samples were further purified using an RNeasy kit (QIAGEN). Real-time reverse transcription PCR (RT-PCR) was performed using DNA Engine Opticon from MJ Research and a QuantiTect SYBR green RT-PCR kit from QIAGEN. Each reaction mixture contained 100 ng of RNA. The reaction mixtures were incubated at 50°C for 30 min for reverse transcription, followed by 15 min at 95°C for heat activation of hot-start Taq polymerase. This was followed by 40 cycles of 15 s at 95°C, 30 s at 55°C (51°C for studies of cro), and 30 s at 72°C. After the last cycle, a melting curve analysis was used to confirm the purity of the product. The PCR products were denatured in a temperature gradient from 50°C to 95°C at 0.03° s–1. The rpnA gene was used as an internal control for each RNA sample. The RNA levels of the target genes were determined using a comparative method (CT method [where CT is cycle threshold]) (27). The data are the results of analysis of at least two independently isolated RNA preparations, each of which was examined by at least three independent real-time RT-PCR runs. For each sample, a non-reverse transcriptase control was included to ensure that there was no contaminating DNA.
Transcription in vitro. PCR was used to recover DNA from PR-GA and PR-GCA (the latter containing the same two mutations as PR-GA and an additional mutation at position –30) for in vitro transcription assays. The isolated fragments extended from an NsiI site in cI (position 37769 in the sequence) to a BglII site in cro (position 38108); the fragment was extended by an additional 8 nucleotides (nt) at the BglII site to include an EcoRI site that was included in the PCR primer. The isolated fragment was then inserted into the dual terminator plasmid pSW305 (53) after cleavage of the plasmid with NsiI and EcoRI restriction endonucleases to create pDF1 (PR-GCA) and pDF2 (PR-GA). Abortive-initiation lag time assays were performed as described previously (53), using HindIII-EcoRI fragments isolated from pDF1, pDF2, and pSW101N (PR+) (53).
Production of transcripts extending to the P22 ant transcription terminator in SW305 were performed as described previously by using an 1,180-bp PvuII-BamHI fragment (53). The terminated transcripts were 290 nt (PR+) or 277 nt (PR-GCA and PR-GA) in length, the difference being due to minor differences in cloning strategy. In the experiment shown in Fig. 5, RNAP (50 nM), substrates, and DNA (2 nM) were added to the reaction mixture simultaneously in the absence of heparin to permit multiple initiations; the reactions were stopped after 20 min.
RESULTS
Identification of a PR mutant unable to grow on a nusA1 host at 32°C. Previous studies have characterized mutations in nus genes that have a temperature-dependent effect on phage production. At higher temperatures, phage yields are dramatically reduced after infection of E. coli nusA1, nusB5, or nusE71 mutants, specifically because of failure of these mutants to support N action (23). However, even at lower temperatures (e.g., 32°C), nusA1 and other nus mutants do not allow development of a number of derivatives (e.g., r32, cIc17, bio256) having more stringent requirements for N-mediated antitermination (46). We refer to the inability of these phage mutants to form plaques on nus mutant strains as the Ssn phenotype (super sensitive to mutant Nus products).
To determine whether the nature of the promoter and the resulting level of transcription can influence subsequent interactions at a NUT site, we screened variants known to be mutated in the PR region to identify those that, in contrast to the wild type, failed to form plaques at 32°C on a lawn formed by a nusA1 mutant. We reasoned that changes in the levels of transcription, presumably down, might influence the effectiveness of N-mediated transcription antitermination. Because the early promoters and operators overlap, we suspected that some mutations in the right operator might also affect the PR promoter. The screen identified one such mutant, which we had obtained as v2v3vs326. This variant, which we will henceforth call vs, contains mutations in the operator region that allow it to replicate in the presence of the cI repressor or high levels of the Cro repressor (42). Based on its failure to form plaques on the nusA1 host at 32°C, vs exhibits the Ssn phenotype. Like other Ssn phages, vs also fails to form plaques on E. coli nusE71 or nusB5 mutants at low temperature (data not shown). Hence, we suspected that the vs mutation or mutations might influence PR activity.
Mutations in the OR/PR region of vs responsible for the Ssn phenotype. DNA sequence analysis identified four nucleotide changes in the OR/PR region of vs, which are located at positions –45, –23, –22, and –14 relative to the PR transcription start site (Fig. 1B). When derivatives were constructed with different combinations of the four mutations (data not shown), we found that two of the changes were necessary and sufficient to confer the Ssn phenotype (Fig. 2A, panel 2), a G-to-A change at –14 (designated G14A) and an A-to-G change at –45 (designated A45G). These mutations are the same as the previously isolated operator mutations NR5 and v3C, respectively (25). We call the derivative containing these mutations PR-GA. This double mutant is unable to produce a phage burst in a nusA1 host at low temperature under conditions in which cI60 (PR+) produces a normal burst of several hundred phage per cell (Fig. 2A).
To gain further insight into the effects of the mutations in vs, we characterized a pseudorevertant, selected for its ability to form plaques on the nusA1 host at low temperature. The pseudorevertant has in addition to the four mutations of vs a change, designated T30C, of T to C at position –30 of PR. To study the effects of this mutation on the Ssn phenotype, this substitution was introduced into PR-GA, generating PR-GCA. PR-GCA produces a substantially larger burst in the nusA1 host at 32°C than does PR-GA (Fig. 2A). By itself, T30C does not significantly affect the phage burst in the nusA1 host (data not shown).
Suppressors of the Ssn phenotype of PR-GA. mutants that express the Ssn phenotype contain mutations that impose a more stringent requirement for N-mediated antitermination in the PR operon (24, 46). That this phenotype results from reduced activity of N at NUTR was demonstrated in several ways. First, mutations affecting other components of the N antitermination complex suppress the Ssn phenotype. Examples of such suppressor mutations are E. coli nusG4 and rpoA(D305E) (47, 50). Second, certain nutR mutations, directly or indirectly, increase the effectiveness of interactions of NUTR RNA with mutant components, such as NusA1 protein, and suppress the Ssn phenotype. For example, the boxA(Con) mutation in nutR suppresses the failure to propagate when infects nusA1 and nusE71 mutant strains at high temperature (24). Third, since N-mediated antitermination is primarily required to allow transcription to transcend transcription terminators in the nin region (Fig. 1A), phage mutations that obviate the effects of these terminators reduce the requirement for NUTR activity (18, 34). Two such mutations that suppress the Ssn phenotype are nin5 (8), a deletion that removes the nin terminators, and Pbyp (3, 29), a promoter formed by mutations in the nin region (6) that allows constitutive expression of Q (Fig. 1A).
We exploited the collection of suppressing mutations to ask whether the Ssn phenotype of PR-GA is due to a defect in N and/or Nus action at NUTR. First, we tested whether nusG4 or rpoA(D305E) suppresses the Ssn phenotype of PR-GA in nusA1 mutant strains. PR-GA propagates significantly better at 32°C on a nusA1 mutant that also contains either the nusG4 or the rpoA(D305E) mutation than it does on the nusA1 parent strain. While the EOP of PR-GA on the nusA1 mutant was <10–4, it was 1 on the nusA1 nusG4 double mutant and 0.3 on the nusA1 rpoA(D305E) double mutant. Second, we constructed derivatives of PR-GA that also contain boxA(Con), nin5, or byp. PR-GA derivatives containing any one of these mutations produce 100 to 1,000 times more phage per burst in the nusA1 host at 32°C than does the parental PR-GA phage (Fig. 2B).
To assess specifically whether the PR-GA promoter reduces the effectiveness of N, we constructed a derivative of PR-GA containing two mutations in the N gene, punA1 and punA133. NpunA1,133, but not wild-type , is efficiently propagated in a nusA1 mutant at 42°C, i.e., the mutant N protein is more effective at forming antitermination complexes than is the wild type (46). As shown in Fig. 2B, panel 2, NpunA1,133 also suppresses the Ssn phenotype caused by the PR-GA mutation in the nusA1 host at 32°C.
Collectively, these data show that all suppressors of other Ssn mutants similarly suppress the Ssn phenotype of PR-GA.
Effect of the PR-GA mutant promoter on antitermination, as measured by transcript level. To determine the effects of A45G and G14A on N-mediated antitermination, we used quantitative real-time RT-PCR to assay mRNA transcribed from sequences in the cro, O, and Q genes during phage infection (Fig. 3A). The levels of mRNA corresponding to these three regions were determined 30 min after infection of strain K95 (nusA1) at 32°C by PR+, PR-GA, and PR-GCA. Values obtained for each region were normalized with respect to those obtained following infection of the nusA+ strain (K37) by PR+ at 32°C.
RNA transcribed from the cro gene (Fig. 3B, panel 1) reflects transcription that is unaffected by N modification and precedes tR1. After infection of K95 (nusA1), the relative cro RNA levels for PR+, PR-GA, and PR-GCA are approximately 0.8, 2.0, and 1.0, respectively. Transcription from the PR-GA promoter is elevated with respect to transcription from wild-type PR because the mutant promoter, like that of the original vs mutant, is at least partially insensitive to Cro protein (data not shown) and is a stronger promoter (see below). The difference between PR-GA and PR-GCA is likely due to the fact that T30C is a mild down mutation, a conclusion based on in vivo studies with other promoters in which that change in sequence causes a decrease in activity by about a factor of two (38; see descriptions of in vitro transcription studies below).
Levels of RNA transcribed from the O gene (Fig. 3B, panel 2) should reflect both the relative rate of transcription through cro and the relative efficiency of transcription antitermination at tR1. In this case, the relative RNA levels for PR+, PR-GA, and PR-GCA are approximately 0.8, 1.0, and 0.7, respectively. Since different primers are used to assay transcription in each region, absolute RNA levels shown in Fig. 3B, panels 1 and 2, cannot be compared directly. However, the difference in the RNA levels (relative to that produced by the wild type) produced after infection by the three phages indicates that the fraction of transcription complexes that are able to proceed through tR1 is reduced in the infection with PR-GA. Based on a comparison of the relative levels of RNA produced by PR-GA and PR+ in the cro region (2.0/0.8 = 2.5) with those produced in the O region (1.0/0.8 = 1.25), we conclude that the mutations in PR-GA caused a reduction of 50% in transcription downstream of terminator tR1. The observed rate of readthrough beyond tR1 is approximately what would be expected in the absence of N antitermination, providing termination readthrough is not affected (7). In contrast, the relative amounts of cro and O RNA produced by the revertant phage PR-GCA are not significantly different from 1. The failure to antiterminate transcription at tR1 is thus a property of PR-GA but not of PR-GCA.
Differences in antitermination properties are far more dramatic when levels of transcript that proceed through the nin terminator region into gene Q are assayed (Fig. 3B, panel 3). If, as is predicted from the genetic experiments and from the data shown in Fig. 3B, panel 2, transcription complexes initiating at PR-GA are not effectively modified by N, a large decrease in Q message should be observed after PR-GA infection of the nusA1 host. This is precisely what was observed. The relative levels of Q RNA after infection by PR+, PR-GA, and PR-GCA were 0.4, 0.02, and 0.6, respectively. The key point is that the level of Q RNA produced by PR-GA was dramatically reduced relative to the amount of RNA produced by either PR+ or PR-GCA, verifying that the Ssn phenotype is due primarily to a failure to antiterminate in the nin region, with a consequent failure to express Q. This in turn would cause a decrease in Q-mediated transcription antitermination from PR' and thus an inhibition of late gene expression.
Transcription in the presence of second-site suppressors. To assess directly whether mutations in that were shown previously to suppress the Ssn phenotype of PR-GA also alleviate the block in transcription of the Q gene, real-time RT-PCR assays were used to measure levels of Q mRNA synthesized by PR-GAbyp (Fig. 3C, panel 1) and PR-GAnin5 (Fig. 3C, panel 2) after infection of the nusA1 host at 32°C. As expected, both byp and nin5 dramatically increase the amount of Q RNA produced by phage also containing the PR-GA mutation. These results show that both the Ssn phenotype of PR-GA and the failure to express Q can be suppressed by deletion of the nin terminators or by creation of a new promoter that bypasses the need for N-mediated antitermination in the nin region. These data provide further support for the idea that the Ssn phenotype of PR-GA is due to failure to antiterminate in the nin region.
Effect of A45G and G14A on promoter activity in vivo. To compare levels of transcription from the variant and wild-type PR promoters, we used a chromosome-based PR transcription reporter system obtained from N. Costantino and D. Court (51). The system is constructed so that lacZ is expressed as a protein fusion to the first 4 codons of cro (Fig. 4). In logarithmically growing nusA+ or nusA1 cells, the PR-GA promoter directs the synthesis of approximately twice as much -galactosidase as does PR+ (Fig. 4). The T30C change in PR-GCA decreases transcription to nearly-wild-type levels. Since Cro protein is not present, -galactosidase levels reflect differences in the rates of transcription directed by the mutant and wild-type PR promoters.
Real-time RT-PCR assays of cro RNA (Fig. 3B, panel 1) reflect transcription from PR in the presence of Cro protein. From data presented herein and published previously (51), Cro has slightly less than a twofold effect on PR transcription. Therefore, differences in levels of expression should reflect differential sensitivity to Cro protein as well as differential promoter activity. On the other hand, -galactosidase produced by cro fusion prophages (Fig. 4) should reflect only differential promoter activity. Thus, the ratio of cro RNA produced by PR-GA to that produced by the wild-type promoter should be greater with the real-time RT-PCR assay than with the cro-lacZ fusion assay. However, the observed ratios of wild-type and mutant promoter activity determined by the two assays were not appreciably different. One possible explanation for the apparent disparity is the difference in gene copy number between the two assays: multicopy following infection in the RT-PCR experiments and a single chromosomal copy by the lacZ assays.
Role of N expression and the Ssn phenotype. Increased transcription from PR should result in increased expression of Cro, which, because of Cro binding at OL, could result in decreased transcription from PL, causing a reduction in N expression. This action of increased Cro levels on N expression offers a possible explanation for the failure of PR-GA to propagate in nus mutants at low temperature. According to this scenario, low levels of N coupled with impaired Nus activity would result in the formation of fewer antitermination complexes and a concomitant increase in unmodified complexes producing the Ssn phenotype of PR-GA.
To assess this possibility, we measured the yield of PR-GA following infection of the nusA1 mutant at 32°C in the presence of N supplied by pJGN (strain K10855). This plasmid has the N gene cloned downstream of Plac as well as a copy of the lacIq gene. Levels of N supplied in the presence of IPTG (isopropyl--D-thiogalactopyranoside) are sufficient to support propagation of Nam7,53, a phage unable to express functional N in the plasmidless parent sup0 nusA1 host at 32°C. At 120 min following infection of the nusA1 pJGN strain with Nam7,53 in the presence of IPTG, there was a burst of 73 phage per infected bacterium. However, under identical conditions following infection with PR-GA, the burst was only 0.35 phage per infected bacterium. Thus, even in the presence of functional levels of N, PR-GA fails to propagate in the nusA1 host at 32°C. This leads us to conclude that a reduction in N expression through the action of increased Cro cannot explain the Ssn phenotype of PR-GA.
Effect of minimal medium on PR-GA growth. One possible explanation for the Ssn phenotype of PR-GA, explored in more detail in Discussion, is that it is a consequence of the increased number of transcription complexes successfully initiating at PR-GA. To test this idea, we determined whether a decrease in RNAP concentration could affect phage production by PR-GA infection of a nusA1 host. The rationale for this experiment is that a decrease in [RNAP] should limit initiation and hence decrease the number of transcription complexes originating at PR-GA and passing through nutR. I. Grigorva and C. Gross (personal communication) found that bacteria grown in minimal medium contain one-fifth as many RNAP molecules per cell as those grown in minimal medium supplemented with Casamino Acids or in a rich medium.
Therefore, we compared phage production in a single round of infection by the PR variants in nusA+ and nusA1 strains at 32°C in minimal M9 maltose medium with phage production in L broth, a rich medium (Fig. 2C). In minimal medium (Fig. 2C, panel 1), the burst of PR+ in the nusA+ strain was about a factor of 10 lower than its burst in L broth (Fig. 2C, panel 2), and the relative decreases in burst size in the nusA1 host were comparable. However, the burst of PR-GA in the nusA1 strain was about three times higher in minimal medium than in L broth. Relative to the effect of growth in minimal medium on PR+ phage production, growth in minimal medium causes approximately a 30-fold increase in burst size of PR-GA after infection of the nusA1 host. Hence, under conditions in which the concentration of RNAP is substantially reduced there is a significant enhancement of PR-GA phage production in the nusA1 host at 32°C.
To determine whether this minimal-medium suppression reflects a general effect on derivatives with weakened N antitermination, we examined the burst of bio256, a derivative that has weakened N antitermination because it expresses a partially defective N protein. Like PR-GA, bio256 fails to propagate in the nusA1 host at 32°C (46). The burst of bio256 in minimal medium at 32°C in the nusA+ host was about 1,000 times greater than it was in the nusA1 host (data not shown). Thus, minimal medium does not reverse the Ssn phenotype of all derivatives with compromised N-NUT systems in nus mutants at 32°C.
Transcription from the mutant promoters in vitro. The observation that -galactosidase synthesis directed by the PR-GA promoter is twice as great as synthesis directed by the wild-type promoter, even in the absence of Cro protein (Fig. 4), suggests that the mutations in PR-GA must affect some aspect of promoter function. Surprisingly, however, the sequence changes in PR-GA (particularly G14A) and PR-GCA (G14A and T30C) reduce agreement of the promoter with consensus sequences (45) and would be predicted to reduce promoter activity relative to that of the wild type.
Therefore, we analyzed activity of wild-type PR and the two mutant promoters kinetically; the rate of synthesis of the initiating trinucleotide, CpApU, was used as a measure of the formation of open complexes (16). The time necessary for open complex formation (obs) at PR-GA was three to four times greater than that at wild-type PR, and the additional mutation in PR-GCA increased obs by an additional factor of 2 to 3 (Table 2).
In general, 1/obs at RNAP concentrations between 30 and 50 nM can be used as a predictor of relative promoter strength in vivo (35). By this measure (as well as by relative values of the overall "on rate," KBkf), wild-type PR should be two to three times as active as PR-GA in vivo. How then can we account for the results shown in Fig. 4 A partial explanation is provided by data obtained with multiround transcription assays. RNAP, promoter-containing DNA, and substrates were added simultaneously to initiate transcription in the absence of heparin, and production of a 277-nt transcript extending to a transcription terminator that reflected transcription initiating at PR was assayed (see Materials and Methods).
Unexpectedly, the level of transcription from PR-GA in these experiments was approximately the same as the level observed for wild-type PR (Fig. 5). Transcription from PR-GCA was reduced by a factor of 2 to 3 relative to the activity of PR-GA, which is consistent with the ability of T30C to suppress the Ssn phenotype, but the reduction relative to the activity of wild-type PR is not as great as would be expected based on the kinetic parameters shown in Table 2.
These data indicate that some event that occurs after open complex formation is affected by the nature of the promoter sequence. In other experiments (data not shown), we found that on a molar basis, approximately 95 to 97% of initiations from PR-GA and PR+ are abortive, producing oligonucleotides approximately 5 to 9 nt in length. Similar results were reported for PR previously (33). Conceivably, the sequence changes in PR-GA alter the frequency of escape from abortive recycling, thereby increasing the frequency of transcription complexes that reach the transcription terminator in vitro (see Discussion).
DISCUSSION
The initial finding that led to our studies was the observation that vs failed to form plaques at low temperature on E. coli nus mutants that impose the Ssn phenotype. Since PR+ is able to form plaques under these conditions, the promoter/operator mutations in some way influence phage development when Nus factor activity is limited. Based on our previous studies with other Ssn phages, we hypothesized that the PR mutations influenced events occurring at NUTR.
This idea was supported by the studies of second-site mutations in both and E. coli strains that were originally identified as suppressors of mutations that reduce the effectiveness of N. We found that these suppressors were also effective in overcoming the failure of PR-GA to produce phage after infection of nusA1 derivatives at 32°C. Furthermore, the A45G and G14A mutations in PR-GA were shown by real-time RT-PCR assays (Fig. 3) to cause a decrease in N-mediated transcription antitermination in vivo. The critical finding was the difference in the relative levels of mRNA produced from regions upstream and downstream of the nin terminator region. These studies demonstrate that transcription complexes initiating at PR-GA differ from those initiating at wild-type PR in the levels of effectiveness with which they are modified by interaction with N and Nus proteins at NUTR.
The locations of genes O and P and the replication origin between tR1 and the nin region (Fig. 1) raise the possibility that the failure of PR-GA to propagate in the nusA1 host at low temperature is due to a defect in replication because of reduced expression of O and P and/or transcription through ori. This possibility is unlikely for the following reasons. First, although readthrough of tR1 when transcription is initiated at PR-GA is only half as efficient as that initiated at PR+, increased transcription from PR-GA means that the overall rates of transcription through O, P, and ori are about the same whether transcription initiates at PR-GA or PR+. Second, the byp mutation, which suppresses the Ssn phenotype of PR-GA and is located downstream from genes encoding the replication machinery, does not affect upstream transcription and therefore is unlikely to have any effect on replication.
Possible mechanisms for promoter influence at NUT sites. We see two ways that an altered promoter sequence could influence downstream events. Changes in the promoter could affect the structure of RNAP, reducing its ability to serve as a substrate for subsequent modification at NUTR. This could result from a change in structure per se or by an effect on the interaction(s) of RNAP with an additional factor(s) during initiation. In this regard, we note that the interaction of 70 with core RNAP can influence subsequent action of RNAP in its -independent mode during elongation (2). Alternatively, the change in the promoter could influence the rate of transcription through the nut site, which, in turn, could influence the effectiveness of the modification process. The addition of T30C to the combination of A45G and G14A, which suppresses the effect of the latter mutations, reduces transcription levels in vivo and in vitro. Moreover, in vivo, the level of transcription from the T30C derivative is similar to that observed with wild-type PR.
We propose that an increase in transcription influences the N modification process because it leads to an increase in the frequency of RNAPs transiting the nut region and thereby results in a relatively high density of RNAPs on the template. Epshtein and colleagues (14, 15) provided in vivo and in vitro evidence supporting a model of transcription in which "robust [frequent] initiation ensures rapid and processive elongation." These workers showed that a trailing RNAP facilitates movement of a leading RNAP through sites that normally slow or stall RNAP movement and that increased concentration of transcribing RNAPs results in an increase in the rate of transcription elongation. Based on these results, we hypothesize that a high density of RNAPs would result in increased rate of movement of the polymerases through the nut region. In this way, the time frame during which each RNAP is properly positioned for modification would be reduced and the efficiency of assembly of the antitermination process would be impaired.
There are two reasons why the assembly of the N antitermination complex would be particularly sensitive to the amount of time available for its formation (that is, the time frame during which RNAP is properly positioned for modification). First, the number of positions along the transcription route at which N can modify RNAP is likely to be extremely limited. Barik et al. (1), in an elegant set of experiments using S30 extracts, showed that N access to RNAP is restricted to those polymerases that are moving through the nut region; those positioned before or after the nut region could not form stable complexes with N. Based on these findings and the structure of the elongation complex, it has been proposed that N binds to nucleotides forming the ascending loop of a NUT BOXB sequence as the RNA is released from the RNA-DNA hybrid in the RNAP prior to formation of the BOXB hairpin structure (20). Second, N modification of RNAP requires interaction of a number of proteins with each other as well as with sites in the NUT RNA (11). In addition to N and NusA, the NusB, NusG, and ribosomal S10 (NusE) proteins are required. Assembly of such a large molecular complex would require several interactions in the time frame during which RNAP is properly positioned for loading. Hence, an increase in the frequency of RNAP transiting nut could drive the polymerases past the modification point at a pace sufficient to reduce the number of RNAPs being modified. This would be particularly noticeable when one of the components of the complex, such as NusA, is partially defective or present in limiting amounts. Although more RNAP molecules would move through the nut region, the number of modified polymerases capable of transcending the downstream nin region of transcription terminators would be substantially reduced. This would result in reduction of Q transcription below the level necessary for late gene expression (32, 54).
Additional support favoring the increased-transcription model is provided by the observation that growth in minimal medium, which is known to reduce the number of RNAP molecules in the cell, also suppresses the effect of the PR-GA mutation on development (Fig. 2C). The reduced number of RNAP molecules (I. Grigorova and C. Gross, personal communication) may lead to a reduction in the frequency of initiation and, in turn, to a reduction in the frequency at which RNAPs pass through the nutR region. In this way, the problem associated with a high density of RNAPs is avoided.
Increased transcription from PR-GA results from both a reduction in binding of Cro protein to OR and the increased activity of the promoter. The two mutations in PR-GA decrease Cro binding to OR, while the cro-lacZ fusion experiments demonstrate that there is increased activity of the promoter even in the absence of cro.
Effects of promoter mutations on transcription initiation. In vitro transcription assays yielded surprising results. The number of completed transcripts (those that terminated at the downstream transcription terminator) did not agree with the relative rate of open complex formation at PR-GA and PR+. These data are unusual in two ways. First, values of obs at 50 nM [RNAP] (the concentration used for transcription assays shown in Fig. 5) can be calculated from the equation given in the footnote to Table 2. These values can be used to predict how many open complexes should form in the first 5 min of incubation. Based on these calculations, even if transcription had been limited to a single round, the number of open complexes formed at wild-type PR should have been 50% greater than the number formed at PR-GA. Because RNAP is in excess in these reactions, the difference between the two promoters should have been more dramatic, since by 2 min of incubation, 80% of the wild-type promoters (but only 35% of the mutant promoters) should have formed open complexes, initiated transcription, and become available for reinitiation. Second, because of the possibility of reinitiation in the absence of heparin, the amount of transcript formed at each promoter should continue to increase substantially during the period from 15 to 30 min. This clearly was not the case.
A possible explanation of both anomalies comes from the work of Kubori and Shimamoto (33), who showed that transcription from a genetically engineered variant of PR yielded primarily (>95%) abortive products approximately 5 to 9 nt long in vitro. Those authors further demonstrated that only one-fourth of open complexes actually produced elongated transcripts and suggested that a large fraction of the abortively recycling complexes were "dead-end" complexes (49), incapable of ever producing a full-length transcript. In similar experiments we also found that on a molar basis >95% of the products produced by both PR and PR-GA were short oligonucleotides. DNA templates in abortively recycling complexes would be unavailable for reinitiation; thus, production of elongated transcripts would not continue indefinitely in our experiments (Fig. 5). We speculate that the mutation(s) in PR-GA increases the fraction of open complexes that escape abortive recycling. Although we did not observe a significant difference between PR and PR-GA in the relative amounts of abortive and full-length (277-nt) transcript (data not shown), a 10 to 20% difference would have been difficult to quantify and could have made a major difference in the production of full-length transcript. This difference would be expected to be exponential, since escape from abortive recycling would permit more-rapid reinitiation.
Clearly, since different naturally occurring promoters abortively recycle at different rates, it would not be unusual for mutations in the promoter to affect abortive recycling (30). Furthermore, mutations in 70 (4, 48) and transcription activation (36) have been shown to affect the production of abortive products.
A change in abortive recycling caused by the mutations may explain the difference between the results in Fig. 5 and those in Table 2, as well as the increased rate of transcription from PR-GA in vivo (Fig. 4). Certainly, differential binding of Cro to wild-type (PR+) and mutant (PR-GA) OR can account for some of the difference between the two promoters in vivo. Even so, the discrepancy between data shown in Fig. 4 (approximately twice as much transcription from PR-GA as from wild-type PR) and the in vitro data is not well understood. However, increasing the concentration of GTP from 5 to 200 μM in vitro produced a 20-fold increase in the amount of abortive product (33). Conceivably, GTP levels could preferentially affect the rate of abortive recycling at wild-type PR, thereby altering the relative activities of PR and PR-GA in vivo.
The phenotype of the PR-GCA promoter suggests that the effect of the mutations in PR-GA on transcription in lac fusion assays is independent of promoter strength per se. T30C decreased promoter strength in vivo by a factor of 2 in a Pant wild-type background (38) and caused only a twofold decrease in the activity of PR-GCA compared to that of PR-GA in the lac fusion assay (Fig. 4). This indicates that PR-GCA retains the unexpectedly high ability to produce elongated transcripts in spite of the fact that its ability to form open complexes has been impaired by the additional (T30C) mutation.
A possible physiological role. Reduction in N-mediated antitermination resulting from increased levels of transcription from PR could play a role in the normal physiology of infection. The delay in Q expression observed with infections has been postulated to extend the time for the decision between lysis and lysogeny. This delay results from a number of factors, including the synthesis of an anti-Q message from cII action at PAQ (28) and a postulated requirement for a threshold level of Q (32, 54). It might be expected that early in infection, when Cro levels have not yet turned down transcription from both PL and PR (26), there would be high levels of transcription from these promoters. High levels of transcription from PR coupled with the levels of N initially expressed early in infection could lead to antitermination at the nin terminators sufficient to allow premature expression of Q and thus reduce the time for the lysis/lysogeny decision. However, low levels of N might also lead to inefficient assembly of N antitermination complexes. This inefficient assembly of antitermination complexes, coupled with increased levels of transcription from PR, may cause failure to read through terminators in nin and thus delayed or reduced Q expression. This mechanism for keeping phage development on the proper time course could be active early in infection, before Cro acts to lower transcription from both early promoters. Reducing Q production through elevated transcription from PR would be another component of the elaborate network of mechanisms acting to ensure that the decision between lysis and lysogeny is effectively regulated.
ACKNOWLEDGMENTS
We thank Christopher LaRock for technical assistance. Nina Costantino and Don Court are thanked for the basic lac fusion used to generate those with promoter variants. Jess Tyler, Jeff Withey, Victor DiRita, and David Gutnick are thanked for critical reading of the manuscript. Theodore Lawrence is thanked for use of the real-time RT-PCR detection system. John Little suggested the possibility that the failure in antitermination complex formation by higher rates of transcription might play a role in the normal physiology of infection. Irina Grigorova and Carol Gross are thanked for allowing us to cite unpublished data.
|| Present address: Division of Math, Science, and Engineering, Northern Virginia Community College, Annandale, VA 22003.
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ABSTRACT
The N protein of phage acts with Escherichia coli Nus proteins at RNA sites, NUT, to modify RNA polymerase (RNAP) to a form that overrides transcription terminators. These interactions have been thought to be the primary determinants of the effectiveness of N-mediated antitermination. We present evidence that the associated promoter, in this case the early PR promoter, can influence N-mediated modification of RNAP even though modification occurs at a site (NUTR) located downstream of the intervening cro gene. As predicted by genetic analysis and confirmed by in vivo transcription studies, a combination of two mutations in PR, at positions –14 and –45 (yielding PR-GA), reduces effectiveness of N modification, while an additional mutation at position –30 (yielding PR-GCA) suppresses this effect. In vivo, the level of PR-GA-directed transcription was twice as great as the wild-type level, while transcription directed by PR-GCA was the same as that directed by the wild-type promoter. However, the rate of open complex formation at PR-GA in vitro was roughly one-third the rate for wild-type PR. We ascribe this apparent discrepancy to an effect of the mutations in PR-GCA on promoter clearance. Based on the in vivo experiments, one plausible explanation for our results is that increased transcription can lead to a failure to form active antitermination complexes with NUT RNA, which, in turn, causes failure to read through downstream termination sites. By blocking antitermination and thus expression of late functions, the effect of increased transcription through nut sites could be physiologically important in maintaining proper regulation of gene expression early in phage development.
INTRODUCTION
Expression of the delayed-early genes of coliphage is regulated by transcription termination and antitermination (19). Transcription initiating at the two early promoters PL and PR partially terminates at terminators tL1 and tR1, respectively (Fig. 1A). Early transcription from PL results in production of N protein, which acts together with a number of host factors (Nus proteins) at NUT sites in nascent-early transcripts with RNA polymerase (RNAP) to form a transcription complex that is resistant to both intrinsic (Rho-independent) and Rho-dependent terminators. Escherichia coli proteins that combine with N and NUT RNA to modify RNAP include NusA, NusB, ribosomal protein S10 (NusE), and NusG (10, 23, 40, 43, 55).
Two classes of mutations in nus genes have been isolated. Class 1 mutations reduce effective N action, while class 2 mutations suppress the action of class 1 mutations by restoring the effectiveness of N in the presence of a class 1 mutation. For example, the failure of E. coli nusA1 or nusE71 mutants to support N action can be suppressed by class 2 mutations in either nusB or nusG (50, 52). In addition to these class 2 nus mutations, a point mutation in rpoA, encoding the subunit of RNAP, suppresses the effects of nusA1 or nusE71 (47).
With the exception of N, all phage-encoded factors required for lytic development of ultimately depend on transcription from PR (21), which is required for expression of cro, cII, replication genes O and P, and the Q gene. Q protein, in turn, is required for the expression of late genes, including those responsible for lysis and morphogenesis (44). The Q protein, like N, is an antitermination protein. However, Q acts through a DNA site, qut, rather than an RNA site, to modify transcription complexes initiating at the late PR' promoter (Fig. 1).
In the absence of N, about 40% of the transcripts initiating at PR transcend tR1 and terminate within the nin terminator region (Fig. 1A) (5, 8). Two mutations that permit to form plaques on nus mutants at high temperature reduce or eliminate the requirement for N-mediated antitermination. These are nin5, a deletion of the nin region, (8) and Pbyp, a mutation generating a new promoter (6) that directs constitutive expression of Q (3, 29). Mutations in the nutR region [e.g., boxA(Con)] and in N (e.g., NpunA1,133) also suppress the effects of nus mutations on phage production (24, 46).
The nut sites are proficient at promoting N-mediated antitermination when placed downstream of promoters other than those of , such as the E. coli gal promoter (12). Thus, in the case of N-mediated antitermination, all of the interactions influencing modification of RNAP to an antiterminating form have been thought to occur at the NUT sites. (Sequences in the RNA are indicated by having every letter capitalized.) In this communication, study of a mutant PR promoter altered by two base changes shows that events occurring at the promoter influence the effectiveness of N modification. Data presented here address two ways that these mutations in PR influence gene expression. First, we demonstrate in vivo that increased transcription observed with the mutant promoter results in failure to modify RNAP at NUTR, which in turn prevents antitermination at downstream terminators in the nin region. Consequently, Q gene expression is not sufficient to allow transcription of late genes. Second, we investigate in vitro how transcription increases in spite of the fact that these mutations in PR decrease the efficiency of open complex formation.
MATERIALS AND METHODS
Bacterial strains and phages. Relevant genotypes and derivations of the E. coli strains and derivatives used in this study are listed in Table 1. Plasmid pJGN was constructed by inserting the N gene in place of the Q gene in pJG100 (31), a plasmid that also has the lacIq gene.
Media and reagents. For most experiments, bacteria were grown in L (Luria) broth. In phage burst experiments, L broth was supplemented with 0.2% maltose. For -galactosidase assays, cells were grown in M9 medium supplemented with 0.2% fructose and 0.1% Casamino Acids. The minimal medium in the phage burst experiments was M9 supplemented with 0.2% maltose. Antibiotics were used at the following concentrations: 50 μg/ml for ampicillin and 30 μg/ml for kanamycin.
The following reagents were purchased from the indicated companies: oligonucleotides (Invitrogen), RNAP holoenzyme (Epicenter), restriction endonucleases (New England BioLabs), Taq polymerase, and RNase free DNase I (Roche Biochemicals).
Phage construction. derivatives with mutations in the PR region were constructed using either of two methods to recombine the DNA with the designed changes into prophages. Both methods exploit the recombination functions of phage to obtain enhanced homologous recombination.
Method A. The procedure developed by Murphy (39) was used to cross designed mutations into the cI857x13 prophage (13) of strain K9227. In this strain, recombination genes are substituted for the recBCD locus and are controlled by the lac promoter.
Method B. The procedure of Court et al. (9) was used to cross designed mutations contained within single-stranded oligonucleotides into the cI857x13 prophage in strain K120. The cI857x13 prophage supplied recombination functions. PR-GA (containing two mutations in PR, at positions –14 and –45) derivatives containing additional mutations, including boxA(Con), byp, and NpunA1,133, were constructed using the method outlined by Oppenheim et al. (41) to cross sequences from single-stranded DNA to the phage genome. PR-GAnin was constructed by recombination between PR-GA and imm434nin with standard phage genetic techniques.
Single-burst and EOP experiments. Production of phage after infection was assayed with single-step growth experiments (17). Efficiency of plating (EOP) was determined by dividing the plaque count on a lawn of the mutant strain by that on a lawn of K37, wild type for nusA, nusG, and rpoD (for details, see reference 22).
Reporter fusion construction. The reporter fusions were constructed by replacing the wild-type PR in a reporter construct with the PR promoter variants. The reporter construct has the cI-PR-cro region of followed by cat-sacB fused to the chromosomal lacZ gene and an adjacent kanR marker (51). The recombineering system of Court et al. (9) was used to generate cro-lacZ fusions under the control of each of the PR derivatives. The constructs were then transduced into nusA+ and nusA1 strains for analysis by use of KanR for selection.
-Galactosidase assays. Bacteria were grown overnight at 32°C in M9 medium supplemented with 0.2% fructose and 0.1% Casamino Acids. Overnight cultures were diluted into the same medium, grown at 32°C to early log phase, and then grown for 1 h at 40°C. Bacteria were harvested, and -galactosidase activity was assayed according to the Miller protocol (37).
Quantitative real-time RT-PCR. Bacteria were grown to mid-log phase in L broth supplemented with 0.2% maltose, concentrated by centrifugation, resuspended in a reduced volume, and incubated with various derivatives on ice at a multiplicity of infection of 5 to allow adsorption. After 20 min, infected bacteria were diluted in L broth supplemented with 10 mM MgSO4 and grown at 32°C for 30 min. Total RNA was prepared from the infected bacteria by use of the TRIzol procedure (Invitrogen). After DNase I digestion, the RNA samples were further purified using an RNeasy kit (QIAGEN). Real-time reverse transcription PCR (RT-PCR) was performed using DNA Engine Opticon from MJ Research and a QuantiTect SYBR green RT-PCR kit from QIAGEN. Each reaction mixture contained 100 ng of RNA. The reaction mixtures were incubated at 50°C for 30 min for reverse transcription, followed by 15 min at 95°C for heat activation of hot-start Taq polymerase. This was followed by 40 cycles of 15 s at 95°C, 30 s at 55°C (51°C for studies of cro), and 30 s at 72°C. After the last cycle, a melting curve analysis was used to confirm the purity of the product. The PCR products were denatured in a temperature gradient from 50°C to 95°C at 0.03° s–1. The rpnA gene was used as an internal control for each RNA sample. The RNA levels of the target genes were determined using a comparative method (CT method [where CT is cycle threshold]) (27). The data are the results of analysis of at least two independently isolated RNA preparations, each of which was examined by at least three independent real-time RT-PCR runs. For each sample, a non-reverse transcriptase control was included to ensure that there was no contaminating DNA.
Transcription in vitro. PCR was used to recover DNA from PR-GA and PR-GCA (the latter containing the same two mutations as PR-GA and an additional mutation at position –30) for in vitro transcription assays. The isolated fragments extended from an NsiI site in cI (position 37769 in the sequence) to a BglII site in cro (position 38108); the fragment was extended by an additional 8 nucleotides (nt) at the BglII site to include an EcoRI site that was included in the PCR primer. The isolated fragment was then inserted into the dual terminator plasmid pSW305 (53) after cleavage of the plasmid with NsiI and EcoRI restriction endonucleases to create pDF1 (PR-GCA) and pDF2 (PR-GA). Abortive-initiation lag time assays were performed as described previously (53), using HindIII-EcoRI fragments isolated from pDF1, pDF2, and pSW101N (PR+) (53).
Production of transcripts extending to the P22 ant transcription terminator in SW305 were performed as described previously by using an 1,180-bp PvuII-BamHI fragment (53). The terminated transcripts were 290 nt (PR+) or 277 nt (PR-GCA and PR-GA) in length, the difference being due to minor differences in cloning strategy. In the experiment shown in Fig. 5, RNAP (50 nM), substrates, and DNA (2 nM) were added to the reaction mixture simultaneously in the absence of heparin to permit multiple initiations; the reactions were stopped after 20 min.
RESULTS
Identification of a PR mutant unable to grow on a nusA1 host at 32°C. Previous studies have characterized mutations in nus genes that have a temperature-dependent effect on phage production. At higher temperatures, phage yields are dramatically reduced after infection of E. coli nusA1, nusB5, or nusE71 mutants, specifically because of failure of these mutants to support N action (23). However, even at lower temperatures (e.g., 32°C), nusA1 and other nus mutants do not allow development of a number of derivatives (e.g., r32, cIc17, bio256) having more stringent requirements for N-mediated antitermination (46). We refer to the inability of these phage mutants to form plaques on nus mutant strains as the Ssn phenotype (super sensitive to mutant Nus products).
To determine whether the nature of the promoter and the resulting level of transcription can influence subsequent interactions at a NUT site, we screened variants known to be mutated in the PR region to identify those that, in contrast to the wild type, failed to form plaques at 32°C on a lawn formed by a nusA1 mutant. We reasoned that changes in the levels of transcription, presumably down, might influence the effectiveness of N-mediated transcription antitermination. Because the early promoters and operators overlap, we suspected that some mutations in the right operator might also affect the PR promoter. The screen identified one such mutant, which we had obtained as v2v3vs326. This variant, which we will henceforth call vs, contains mutations in the operator region that allow it to replicate in the presence of the cI repressor or high levels of the Cro repressor (42). Based on its failure to form plaques on the nusA1 host at 32°C, vs exhibits the Ssn phenotype. Like other Ssn phages, vs also fails to form plaques on E. coli nusE71 or nusB5 mutants at low temperature (data not shown). Hence, we suspected that the vs mutation or mutations might influence PR activity.
Mutations in the OR/PR region of vs responsible for the Ssn phenotype. DNA sequence analysis identified four nucleotide changes in the OR/PR region of vs, which are located at positions –45, –23, –22, and –14 relative to the PR transcription start site (Fig. 1B). When derivatives were constructed with different combinations of the four mutations (data not shown), we found that two of the changes were necessary and sufficient to confer the Ssn phenotype (Fig. 2A, panel 2), a G-to-A change at –14 (designated G14A) and an A-to-G change at –45 (designated A45G). These mutations are the same as the previously isolated operator mutations NR5 and v3C, respectively (25). We call the derivative containing these mutations PR-GA. This double mutant is unable to produce a phage burst in a nusA1 host at low temperature under conditions in which cI60 (PR+) produces a normal burst of several hundred phage per cell (Fig. 2A).
To gain further insight into the effects of the mutations in vs, we characterized a pseudorevertant, selected for its ability to form plaques on the nusA1 host at low temperature. The pseudorevertant has in addition to the four mutations of vs a change, designated T30C, of T to C at position –30 of PR. To study the effects of this mutation on the Ssn phenotype, this substitution was introduced into PR-GA, generating PR-GCA. PR-GCA produces a substantially larger burst in the nusA1 host at 32°C than does PR-GA (Fig. 2A). By itself, T30C does not significantly affect the phage burst in the nusA1 host (data not shown).
Suppressors of the Ssn phenotype of PR-GA. mutants that express the Ssn phenotype contain mutations that impose a more stringent requirement for N-mediated antitermination in the PR operon (24, 46). That this phenotype results from reduced activity of N at NUTR was demonstrated in several ways. First, mutations affecting other components of the N antitermination complex suppress the Ssn phenotype. Examples of such suppressor mutations are E. coli nusG4 and rpoA(D305E) (47, 50). Second, certain nutR mutations, directly or indirectly, increase the effectiveness of interactions of NUTR RNA with mutant components, such as NusA1 protein, and suppress the Ssn phenotype. For example, the boxA(Con) mutation in nutR suppresses the failure to propagate when infects nusA1 and nusE71 mutant strains at high temperature (24). Third, since N-mediated antitermination is primarily required to allow transcription to transcend transcription terminators in the nin region (Fig. 1A), phage mutations that obviate the effects of these terminators reduce the requirement for NUTR activity (18, 34). Two such mutations that suppress the Ssn phenotype are nin5 (8), a deletion that removes the nin terminators, and Pbyp (3, 29), a promoter formed by mutations in the nin region (6) that allows constitutive expression of Q (Fig. 1A).
We exploited the collection of suppressing mutations to ask whether the Ssn phenotype of PR-GA is due to a defect in N and/or Nus action at NUTR. First, we tested whether nusG4 or rpoA(D305E) suppresses the Ssn phenotype of PR-GA in nusA1 mutant strains. PR-GA propagates significantly better at 32°C on a nusA1 mutant that also contains either the nusG4 or the rpoA(D305E) mutation than it does on the nusA1 parent strain. While the EOP of PR-GA on the nusA1 mutant was <10–4, it was 1 on the nusA1 nusG4 double mutant and 0.3 on the nusA1 rpoA(D305E) double mutant. Second, we constructed derivatives of PR-GA that also contain boxA(Con), nin5, or byp. PR-GA derivatives containing any one of these mutations produce 100 to 1,000 times more phage per burst in the nusA1 host at 32°C than does the parental PR-GA phage (Fig. 2B).
To assess specifically whether the PR-GA promoter reduces the effectiveness of N, we constructed a derivative of PR-GA containing two mutations in the N gene, punA1 and punA133. NpunA1,133, but not wild-type , is efficiently propagated in a nusA1 mutant at 42°C, i.e., the mutant N protein is more effective at forming antitermination complexes than is the wild type (46). As shown in Fig. 2B, panel 2, NpunA1,133 also suppresses the Ssn phenotype caused by the PR-GA mutation in the nusA1 host at 32°C.
Collectively, these data show that all suppressors of other Ssn mutants similarly suppress the Ssn phenotype of PR-GA.
Effect of the PR-GA mutant promoter on antitermination, as measured by transcript level. To determine the effects of A45G and G14A on N-mediated antitermination, we used quantitative real-time RT-PCR to assay mRNA transcribed from sequences in the cro, O, and Q genes during phage infection (Fig. 3A). The levels of mRNA corresponding to these three regions were determined 30 min after infection of strain K95 (nusA1) at 32°C by PR+, PR-GA, and PR-GCA. Values obtained for each region were normalized with respect to those obtained following infection of the nusA+ strain (K37) by PR+ at 32°C.
RNA transcribed from the cro gene (Fig. 3B, panel 1) reflects transcription that is unaffected by N modification and precedes tR1. After infection of K95 (nusA1), the relative cro RNA levels for PR+, PR-GA, and PR-GCA are approximately 0.8, 2.0, and 1.0, respectively. Transcription from the PR-GA promoter is elevated with respect to transcription from wild-type PR because the mutant promoter, like that of the original vs mutant, is at least partially insensitive to Cro protein (data not shown) and is a stronger promoter (see below). The difference between PR-GA and PR-GCA is likely due to the fact that T30C is a mild down mutation, a conclusion based on in vivo studies with other promoters in which that change in sequence causes a decrease in activity by about a factor of two (38; see descriptions of in vitro transcription studies below).
Levels of RNA transcribed from the O gene (Fig. 3B, panel 2) should reflect both the relative rate of transcription through cro and the relative efficiency of transcription antitermination at tR1. In this case, the relative RNA levels for PR+, PR-GA, and PR-GCA are approximately 0.8, 1.0, and 0.7, respectively. Since different primers are used to assay transcription in each region, absolute RNA levels shown in Fig. 3B, panels 1 and 2, cannot be compared directly. However, the difference in the RNA levels (relative to that produced by the wild type) produced after infection by the three phages indicates that the fraction of transcription complexes that are able to proceed through tR1 is reduced in the infection with PR-GA. Based on a comparison of the relative levels of RNA produced by PR-GA and PR+ in the cro region (2.0/0.8 = 2.5) with those produced in the O region (1.0/0.8 = 1.25), we conclude that the mutations in PR-GA caused a reduction of 50% in transcription downstream of terminator tR1. The observed rate of readthrough beyond tR1 is approximately what would be expected in the absence of N antitermination, providing termination readthrough is not affected (7). In contrast, the relative amounts of cro and O RNA produced by the revertant phage PR-GCA are not significantly different from 1. The failure to antiterminate transcription at tR1 is thus a property of PR-GA but not of PR-GCA.
Differences in antitermination properties are far more dramatic when levels of transcript that proceed through the nin terminator region into gene Q are assayed (Fig. 3B, panel 3). If, as is predicted from the genetic experiments and from the data shown in Fig. 3B, panel 2, transcription complexes initiating at PR-GA are not effectively modified by N, a large decrease in Q message should be observed after PR-GA infection of the nusA1 host. This is precisely what was observed. The relative levels of Q RNA after infection by PR+, PR-GA, and PR-GCA were 0.4, 0.02, and 0.6, respectively. The key point is that the level of Q RNA produced by PR-GA was dramatically reduced relative to the amount of RNA produced by either PR+ or PR-GCA, verifying that the Ssn phenotype is due primarily to a failure to antiterminate in the nin region, with a consequent failure to express Q. This in turn would cause a decrease in Q-mediated transcription antitermination from PR' and thus an inhibition of late gene expression.
Transcription in the presence of second-site suppressors. To assess directly whether mutations in that were shown previously to suppress the Ssn phenotype of PR-GA also alleviate the block in transcription of the Q gene, real-time RT-PCR assays were used to measure levels of Q mRNA synthesized by PR-GAbyp (Fig. 3C, panel 1) and PR-GAnin5 (Fig. 3C, panel 2) after infection of the nusA1 host at 32°C. As expected, both byp and nin5 dramatically increase the amount of Q RNA produced by phage also containing the PR-GA mutation. These results show that both the Ssn phenotype of PR-GA and the failure to express Q can be suppressed by deletion of the nin terminators or by creation of a new promoter that bypasses the need for N-mediated antitermination in the nin region. These data provide further support for the idea that the Ssn phenotype of PR-GA is due to failure to antiterminate in the nin region.
Effect of A45G and G14A on promoter activity in vivo. To compare levels of transcription from the variant and wild-type PR promoters, we used a chromosome-based PR transcription reporter system obtained from N. Costantino and D. Court (51). The system is constructed so that lacZ is expressed as a protein fusion to the first 4 codons of cro (Fig. 4). In logarithmically growing nusA+ or nusA1 cells, the PR-GA promoter directs the synthesis of approximately twice as much -galactosidase as does PR+ (Fig. 4). The T30C change in PR-GCA decreases transcription to nearly-wild-type levels. Since Cro protein is not present, -galactosidase levels reflect differences in the rates of transcription directed by the mutant and wild-type PR promoters.
Real-time RT-PCR assays of cro RNA (Fig. 3B, panel 1) reflect transcription from PR in the presence of Cro protein. From data presented herein and published previously (51), Cro has slightly less than a twofold effect on PR transcription. Therefore, differences in levels of expression should reflect differential sensitivity to Cro protein as well as differential promoter activity. On the other hand, -galactosidase produced by cro fusion prophages (Fig. 4) should reflect only differential promoter activity. Thus, the ratio of cro RNA produced by PR-GA to that produced by the wild-type promoter should be greater with the real-time RT-PCR assay than with the cro-lacZ fusion assay. However, the observed ratios of wild-type and mutant promoter activity determined by the two assays were not appreciably different. One possible explanation for the apparent disparity is the difference in gene copy number between the two assays: multicopy following infection in the RT-PCR experiments and a single chromosomal copy by the lacZ assays.
Role of N expression and the Ssn phenotype. Increased transcription from PR should result in increased expression of Cro, which, because of Cro binding at OL, could result in decreased transcription from PL, causing a reduction in N expression. This action of increased Cro levels on N expression offers a possible explanation for the failure of PR-GA to propagate in nus mutants at low temperature. According to this scenario, low levels of N coupled with impaired Nus activity would result in the formation of fewer antitermination complexes and a concomitant increase in unmodified complexes producing the Ssn phenotype of PR-GA.
To assess this possibility, we measured the yield of PR-GA following infection of the nusA1 mutant at 32°C in the presence of N supplied by pJGN (strain K10855). This plasmid has the N gene cloned downstream of Plac as well as a copy of the lacIq gene. Levels of N supplied in the presence of IPTG (isopropyl--D-thiogalactopyranoside) are sufficient to support propagation of Nam7,53, a phage unable to express functional N in the plasmidless parent sup0 nusA1 host at 32°C. At 120 min following infection of the nusA1 pJGN strain with Nam7,53 in the presence of IPTG, there was a burst of 73 phage per infected bacterium. However, under identical conditions following infection with PR-GA, the burst was only 0.35 phage per infected bacterium. Thus, even in the presence of functional levels of N, PR-GA fails to propagate in the nusA1 host at 32°C. This leads us to conclude that a reduction in N expression through the action of increased Cro cannot explain the Ssn phenotype of PR-GA.
Effect of minimal medium on PR-GA growth. One possible explanation for the Ssn phenotype of PR-GA, explored in more detail in Discussion, is that it is a consequence of the increased number of transcription complexes successfully initiating at PR-GA. To test this idea, we determined whether a decrease in RNAP concentration could affect phage production by PR-GA infection of a nusA1 host. The rationale for this experiment is that a decrease in [RNAP] should limit initiation and hence decrease the number of transcription complexes originating at PR-GA and passing through nutR. I. Grigorva and C. Gross (personal communication) found that bacteria grown in minimal medium contain one-fifth as many RNAP molecules per cell as those grown in minimal medium supplemented with Casamino Acids or in a rich medium.
Therefore, we compared phage production in a single round of infection by the PR variants in nusA+ and nusA1 strains at 32°C in minimal M9 maltose medium with phage production in L broth, a rich medium (Fig. 2C). In minimal medium (Fig. 2C, panel 1), the burst of PR+ in the nusA+ strain was about a factor of 10 lower than its burst in L broth (Fig. 2C, panel 2), and the relative decreases in burst size in the nusA1 host were comparable. However, the burst of PR-GA in the nusA1 strain was about three times higher in minimal medium than in L broth. Relative to the effect of growth in minimal medium on PR+ phage production, growth in minimal medium causes approximately a 30-fold increase in burst size of PR-GA after infection of the nusA1 host. Hence, under conditions in which the concentration of RNAP is substantially reduced there is a significant enhancement of PR-GA phage production in the nusA1 host at 32°C.
To determine whether this minimal-medium suppression reflects a general effect on derivatives with weakened N antitermination, we examined the burst of bio256, a derivative that has weakened N antitermination because it expresses a partially defective N protein. Like PR-GA, bio256 fails to propagate in the nusA1 host at 32°C (46). The burst of bio256 in minimal medium at 32°C in the nusA+ host was about 1,000 times greater than it was in the nusA1 host (data not shown). Thus, minimal medium does not reverse the Ssn phenotype of all derivatives with compromised N-NUT systems in nus mutants at 32°C.
Transcription from the mutant promoters in vitro. The observation that -galactosidase synthesis directed by the PR-GA promoter is twice as great as synthesis directed by the wild-type promoter, even in the absence of Cro protein (Fig. 4), suggests that the mutations in PR-GA must affect some aspect of promoter function. Surprisingly, however, the sequence changes in PR-GA (particularly G14A) and PR-GCA (G14A and T30C) reduce agreement of the promoter with consensus sequences (45) and would be predicted to reduce promoter activity relative to that of the wild type.
Therefore, we analyzed activity of wild-type PR and the two mutant promoters kinetically; the rate of synthesis of the initiating trinucleotide, CpApU, was used as a measure of the formation of open complexes (16). The time necessary for open complex formation (obs) at PR-GA was three to four times greater than that at wild-type PR, and the additional mutation in PR-GCA increased obs by an additional factor of 2 to 3 (Table 2).
In general, 1/obs at RNAP concentrations between 30 and 50 nM can be used as a predictor of relative promoter strength in vivo (35). By this measure (as well as by relative values of the overall "on rate," KBkf), wild-type PR should be two to three times as active as PR-GA in vivo. How then can we account for the results shown in Fig. 4 A partial explanation is provided by data obtained with multiround transcription assays. RNAP, promoter-containing DNA, and substrates were added simultaneously to initiate transcription in the absence of heparin, and production of a 277-nt transcript extending to a transcription terminator that reflected transcription initiating at PR was assayed (see Materials and Methods).
Unexpectedly, the level of transcription from PR-GA in these experiments was approximately the same as the level observed for wild-type PR (Fig. 5). Transcription from PR-GCA was reduced by a factor of 2 to 3 relative to the activity of PR-GA, which is consistent with the ability of T30C to suppress the Ssn phenotype, but the reduction relative to the activity of wild-type PR is not as great as would be expected based on the kinetic parameters shown in Table 2.
These data indicate that some event that occurs after open complex formation is affected by the nature of the promoter sequence. In other experiments (data not shown), we found that on a molar basis, approximately 95 to 97% of initiations from PR-GA and PR+ are abortive, producing oligonucleotides approximately 5 to 9 nt in length. Similar results were reported for PR previously (33). Conceivably, the sequence changes in PR-GA alter the frequency of escape from abortive recycling, thereby increasing the frequency of transcription complexes that reach the transcription terminator in vitro (see Discussion).
DISCUSSION
The initial finding that led to our studies was the observation that vs failed to form plaques at low temperature on E. coli nus mutants that impose the Ssn phenotype. Since PR+ is able to form plaques under these conditions, the promoter/operator mutations in some way influence phage development when Nus factor activity is limited. Based on our previous studies with other Ssn phages, we hypothesized that the PR mutations influenced events occurring at NUTR.
This idea was supported by the studies of second-site mutations in both and E. coli strains that were originally identified as suppressors of mutations that reduce the effectiveness of N. We found that these suppressors were also effective in overcoming the failure of PR-GA to produce phage after infection of nusA1 derivatives at 32°C. Furthermore, the A45G and G14A mutations in PR-GA were shown by real-time RT-PCR assays (Fig. 3) to cause a decrease in N-mediated transcription antitermination in vivo. The critical finding was the difference in the relative levels of mRNA produced from regions upstream and downstream of the nin terminator region. These studies demonstrate that transcription complexes initiating at PR-GA differ from those initiating at wild-type PR in the levels of effectiveness with which they are modified by interaction with N and Nus proteins at NUTR.
The locations of genes O and P and the replication origin between tR1 and the nin region (Fig. 1) raise the possibility that the failure of PR-GA to propagate in the nusA1 host at low temperature is due to a defect in replication because of reduced expression of O and P and/or transcription through ori. This possibility is unlikely for the following reasons. First, although readthrough of tR1 when transcription is initiated at PR-GA is only half as efficient as that initiated at PR+, increased transcription from PR-GA means that the overall rates of transcription through O, P, and ori are about the same whether transcription initiates at PR-GA or PR+. Second, the byp mutation, which suppresses the Ssn phenotype of PR-GA and is located downstream from genes encoding the replication machinery, does not affect upstream transcription and therefore is unlikely to have any effect on replication.
Possible mechanisms for promoter influence at NUT sites. We see two ways that an altered promoter sequence could influence downstream events. Changes in the promoter could affect the structure of RNAP, reducing its ability to serve as a substrate for subsequent modification at NUTR. This could result from a change in structure per se or by an effect on the interaction(s) of RNAP with an additional factor(s) during initiation. In this regard, we note that the interaction of 70 with core RNAP can influence subsequent action of RNAP in its -independent mode during elongation (2). Alternatively, the change in the promoter could influence the rate of transcription through the nut site, which, in turn, could influence the effectiveness of the modification process. The addition of T30C to the combination of A45G and G14A, which suppresses the effect of the latter mutations, reduces transcription levels in vivo and in vitro. Moreover, in vivo, the level of transcription from the T30C derivative is similar to that observed with wild-type PR.
We propose that an increase in transcription influences the N modification process because it leads to an increase in the frequency of RNAPs transiting the nut region and thereby results in a relatively high density of RNAPs on the template. Epshtein and colleagues (14, 15) provided in vivo and in vitro evidence supporting a model of transcription in which "robust [frequent] initiation ensures rapid and processive elongation." These workers showed that a trailing RNAP facilitates movement of a leading RNAP through sites that normally slow or stall RNAP movement and that increased concentration of transcribing RNAPs results in an increase in the rate of transcription elongation. Based on these results, we hypothesize that a high density of RNAPs would result in increased rate of movement of the polymerases through the nut region. In this way, the time frame during which each RNAP is properly positioned for modification would be reduced and the efficiency of assembly of the antitermination process would be impaired.
There are two reasons why the assembly of the N antitermination complex would be particularly sensitive to the amount of time available for its formation (that is, the time frame during which RNAP is properly positioned for modification). First, the number of positions along the transcription route at which N can modify RNAP is likely to be extremely limited. Barik et al. (1), in an elegant set of experiments using S30 extracts, showed that N access to RNAP is restricted to those polymerases that are moving through the nut region; those positioned before or after the nut region could not form stable complexes with N. Based on these findings and the structure of the elongation complex, it has been proposed that N binds to nucleotides forming the ascending loop of a NUT BOXB sequence as the RNA is released from the RNA-DNA hybrid in the RNAP prior to formation of the BOXB hairpin structure (20). Second, N modification of RNAP requires interaction of a number of proteins with each other as well as with sites in the NUT RNA (11). In addition to N and NusA, the NusB, NusG, and ribosomal S10 (NusE) proteins are required. Assembly of such a large molecular complex would require several interactions in the time frame during which RNAP is properly positioned for loading. Hence, an increase in the frequency of RNAP transiting nut could drive the polymerases past the modification point at a pace sufficient to reduce the number of RNAPs being modified. This would be particularly noticeable when one of the components of the complex, such as NusA, is partially defective or present in limiting amounts. Although more RNAP molecules would move through the nut region, the number of modified polymerases capable of transcending the downstream nin region of transcription terminators would be substantially reduced. This would result in reduction of Q transcription below the level necessary for late gene expression (32, 54).
Additional support favoring the increased-transcription model is provided by the observation that growth in minimal medium, which is known to reduce the number of RNAP molecules in the cell, also suppresses the effect of the PR-GA mutation on development (Fig. 2C). The reduced number of RNAP molecules (I. Grigorova and C. Gross, personal communication) may lead to a reduction in the frequency of initiation and, in turn, to a reduction in the frequency at which RNAPs pass through the nutR region. In this way, the problem associated with a high density of RNAPs is avoided.
Increased transcription from PR-GA results from both a reduction in binding of Cro protein to OR and the increased activity of the promoter. The two mutations in PR-GA decrease Cro binding to OR, while the cro-lacZ fusion experiments demonstrate that there is increased activity of the promoter even in the absence of cro.
Effects of promoter mutations on transcription initiation. In vitro transcription assays yielded surprising results. The number of completed transcripts (those that terminated at the downstream transcription terminator) did not agree with the relative rate of open complex formation at PR-GA and PR+. These data are unusual in two ways. First, values of obs at 50 nM [RNAP] (the concentration used for transcription assays shown in Fig. 5) can be calculated from the equation given in the footnote to Table 2. These values can be used to predict how many open complexes should form in the first 5 min of incubation. Based on these calculations, even if transcription had been limited to a single round, the number of open complexes formed at wild-type PR should have been 50% greater than the number formed at PR-GA. Because RNAP is in excess in these reactions, the difference between the two promoters should have been more dramatic, since by 2 min of incubation, 80% of the wild-type promoters (but only 35% of the mutant promoters) should have formed open complexes, initiated transcription, and become available for reinitiation. Second, because of the possibility of reinitiation in the absence of heparin, the amount of transcript formed at each promoter should continue to increase substantially during the period from 15 to 30 min. This clearly was not the case.
A possible explanation of both anomalies comes from the work of Kubori and Shimamoto (33), who showed that transcription from a genetically engineered variant of PR yielded primarily (>95%) abortive products approximately 5 to 9 nt long in vitro. Those authors further demonstrated that only one-fourth of open complexes actually produced elongated transcripts and suggested that a large fraction of the abortively recycling complexes were "dead-end" complexes (49), incapable of ever producing a full-length transcript. In similar experiments we also found that on a molar basis >95% of the products produced by both PR and PR-GA were short oligonucleotides. DNA templates in abortively recycling complexes would be unavailable for reinitiation; thus, production of elongated transcripts would not continue indefinitely in our experiments (Fig. 5). We speculate that the mutation(s) in PR-GA increases the fraction of open complexes that escape abortive recycling. Although we did not observe a significant difference between PR and PR-GA in the relative amounts of abortive and full-length (277-nt) transcript (data not shown), a 10 to 20% difference would have been difficult to quantify and could have made a major difference in the production of full-length transcript. This difference would be expected to be exponential, since escape from abortive recycling would permit more-rapid reinitiation.
Clearly, since different naturally occurring promoters abortively recycle at different rates, it would not be unusual for mutations in the promoter to affect abortive recycling (30). Furthermore, mutations in 70 (4, 48) and transcription activation (36) have been shown to affect the production of abortive products.
A change in abortive recycling caused by the mutations may explain the difference between the results in Fig. 5 and those in Table 2, as well as the increased rate of transcription from PR-GA in vivo (Fig. 4). Certainly, differential binding of Cro to wild-type (PR+) and mutant (PR-GA) OR can account for some of the difference between the two promoters in vivo. Even so, the discrepancy between data shown in Fig. 4 (approximately twice as much transcription from PR-GA as from wild-type PR) and the in vitro data is not well understood. However, increasing the concentration of GTP from 5 to 200 μM in vitro produced a 20-fold increase in the amount of abortive product (33). Conceivably, GTP levels could preferentially affect the rate of abortive recycling at wild-type PR, thereby altering the relative activities of PR and PR-GA in vivo.
The phenotype of the PR-GCA promoter suggests that the effect of the mutations in PR-GA on transcription in lac fusion assays is independent of promoter strength per se. T30C decreased promoter strength in vivo by a factor of 2 in a Pant wild-type background (38) and caused only a twofold decrease in the activity of PR-GCA compared to that of PR-GA in the lac fusion assay (Fig. 4). This indicates that PR-GCA retains the unexpectedly high ability to produce elongated transcripts in spite of the fact that its ability to form open complexes has been impaired by the additional (T30C) mutation.
A possible physiological role. Reduction in N-mediated antitermination resulting from increased levels of transcription from PR could play a role in the normal physiology of infection. The delay in Q expression observed with infections has been postulated to extend the time for the decision between lysis and lysogeny. This delay results from a number of factors, including the synthesis of an anti-Q message from cII action at PAQ (28) and a postulated requirement for a threshold level of Q (32, 54). It might be expected that early in infection, when Cro levels have not yet turned down transcription from both PL and PR (26), there would be high levels of transcription from these promoters. High levels of transcription from PR coupled with the levels of N initially expressed early in infection could lead to antitermination at the nin terminators sufficient to allow premature expression of Q and thus reduce the time for the lysis/lysogeny decision. However, low levels of N might also lead to inefficient assembly of N antitermination complexes. This inefficient assembly of antitermination complexes, coupled with increased levels of transcription from PR, may cause failure to read through terminators in nin and thus delayed or reduced Q expression. This mechanism for keeping phage development on the proper time course could be active early in infection, before Cro acts to lower transcription from both early promoters. Reducing Q production through elevated transcription from PR would be another component of the elaborate network of mechanisms acting to ensure that the decision between lysis and lysogeny is effectively regulated.
ACKNOWLEDGMENTS
We thank Christopher LaRock for technical assistance. Nina Costantino and Don Court are thanked for the basic lac fusion used to generate those with promoter variants. Jess Tyler, Jeff Withey, Victor DiRita, and David Gutnick are thanked for critical reading of the manuscript. Theodore Lawrence is thanked for use of the real-time RT-PCR detection system. John Little suggested the possibility that the failure in antitermination complex formation by higher rates of transcription might play a role in the normal physiology of infection. Irina Grigorova and Carol Gross are thanked for allowing us to cite unpublished data.
|| Present address: Division of Math, Science, and Engineering, Northern Virginia Community College, Annandale, VA 22003.
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