Variable and conserved elements of human ribosomes surrounding the mRN
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《核酸研究医学期刊》
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva, 8, Novosibirsk, 630090, Russia
* To whom correspondence should be addressed. Tel: +3832 35 62 29; Fax: +3832 33 36 77; Email: karpova@niboch.nsc.ru
ABSTRACT
This study is centred upon an important biological problem concerning the structural organization of mammalian ribosomes that cannot be studied by X-ray analysis because 80S ribosome crystals are still unavailable. Here, positioning of the mRNA on 80S ribosomes was studied using mRNA analogues containing the perfluorophenylazide cross-linker on either the guanosine or an uridine residue. The modi-fied nucleotides were directed to positions from –9 to +6 with respect to the first nucleotide of the P site bound codon by a tRNA cognate to the triplet targeted to the P site. Upon mild UV-irradiation, the modified nucleotides at positions +4 to +6 cross-linked to protein S15 and 18S rRNA nucleotides A1823–A1825. In addition, modified guanosines in positions +5 and +6 also cross-linked to G626, and in position +1 to G1702. Cross-linking from the upstream positions was mainly to protein S26 that has no prokaryotic homologues. These findings indicate that the tail of mammalian S15 comes closer to the decoding site than that of its prokaryotic homologue S19, and that the environments of the upstream part of mRNA on 80S and 70S ribosomes differ. On the other hand, the results confirm the widely accepted idea regarding the conserved nature of the decoding site of the small subunit rRNA.
INTRODUCTION
Genetic information is transmitted to the ribosome where trinucleotide codons of the mRNA are recognized by anticodons of the cognate tRNAs at the decoding site. This function is common to prokaryotic and eukaryotic ribosomes. The structure of the decoding site of the prokaryotic ribosome and the molecular basis for decoding are known today at the atomic level thanks to the remarkable progress of X-ray crystallographic analysis (1–4). It is generally assumed that arrangement of the mRNA on the eukaryotic ribosome resembles that on the prokaryotic ribosome. Nevertheless, little is known about the structure of the mRNA-binding site of eukaryotic ribosomes, because many methods used to study prokaryotic ribosomes are not applicable to eukaryotic ribosomes yet. For instance, still crystals of eukaryotic ribosomal subunits suitable for X-ray analysis are not available. Approaches based on reconstitution of functionally active eukaryotic ribosomal subunits from ribosomal proteins and rRNAs also are not applicable to study eukaryotic ribosomes since to date no methodology is available for the reconstitution of the eukaryotic ribosomal subunits in vitro. One approach that can provide detailed information on the structures of functional sites of eukaryotic ribosomes is site-directed cross-linking. The method is based on the use of mRNA analogues that contain chemically reactive groups at defined positions . This makes it possible to determine the ribosomal components contacting—or located close to—the modified mRNA nucleotides. The majority of the data on positioning of the mRNA on the prokaryotic ribosome obtained by means of the site-directed cross-linking approach turned out to be in general agreement with recent X-ray crystallographic data (1–4). This justifies the use of cross-linking approach to study functional sites of eukaryotic ribosomes.
In earlier studies using s4U-containing mRNA analogues, the substantial differences in arrangement of mRNA on human and Escherichia coli ribosomes have been found. In particular, these mRNA analogues produced much fewer numbers of cross-links with 18S rRNA in the 80S ribosome than with 16S rRNA in the 70S ribosome (6,7). It was suggested that the mRNA track is stricter in mammalian ribosomes than in eubacterial ribosomes, or that internal flexibility of mammalian ribosomes is lower than that of eubacterial ribosomes (7). A very limited number of cross-links of s4U-containing mRNA analogues to 18S rRNA in human ribosomes might also be due to other reasons. Cross-links would not occur if the mRNA nucleotide contacts the 18S rRNA by its ribose-phosphate backbone, or if the contacting mRNA and rRNA bases were in mutual orientations not suitable for the cross-linking. In addition, large portions of the mRNA on the human ribosome might be in contact with proteins rather than with the rRNA. In any event, these data indicate that the mRNA-binding track is arranged differently on prokaryotic than on eukaryotic ribosomes. These differences can be studied more extensively by means of mRNA analogues with conformationally flexible cross-linkers capable of reaching any ribosomal component in the vicinity of the modified mRNA nucleotide.
Short mRNA analogues, derivatives of oligoribonucleotides (from hexa- to dodecamers) that contain a perfluoroarylazide cross-linker at different locations, turned out to be suitable tools for studing mRNA-binding site of eukaryotic (in particular, human) ribosomes (8–12). These templates may be positioned exactly on the 80S ribosome by the addition of tRNA cognate to the selected mRNA codon targeted to the P site, since short oligoribonucleotides barely bind to the ribosomes in the absence of tRNA (9,10,12). Positioning of short mRNA analogues on the 80S ribosome is functionally correct because the ribosome is capable of translating such analogues to form oligopeptides encoded by the mRNA sequences (12). Perfluorophenylazide groups attached to the selected nucleotides of the mRNA analogue via conformationally flexible spacers are appropriate cross-linkers capable of effective attachment to rRNA as well as to proteins in the ribosome (12). Using a set of short mRNA analogues bearing the cross-linker at either the N7 atom of the guanosine or at the 5'-terminal nucleotide (either to the 5'-phosphate or C5 atom of uridine), we have determined 18S rRNA nucleotides and ribosomal proteins neighbouring the P and E site bound codons and the first nucleotide of the A site bound codon on human 80S ribosome (10,11,13). For the cross-linker at mRNA positions +1 and –3 with respect to the first nucleotide of the P site bound codon, the target for modification in the 80S ribosome was sensitive to the nature of the nucleotide that bore the cross-linker (11,13,14). Evidently, the application of a set of mRNA analogues in which the nature of the nucleotides carrying the photoactivatable groups varies, provides more comprehensive information on the environment of mRNA nucleotides on the ribosome.
In the present study, to determine the 18S rRNA nucleotides neighbouring first nucleotide of the P site bound codon, a derivative of the hexaribonucleotide UUUGUU (containing the triplets UUU and GUU coding for Phe and Val, respectively) with a perfluorophenylazide group at the N7 atom of the guanosine was used. To reveal 18S rRNA nucleotides and ribosomal proteins in the vicinity of the A site bound codon, a set of oligomers (from hexa- to nonamers) with a perfluorophenylazide group at either the N7 atom of the guanosine or the C5 atom of the uridine was used. Ribosomal components that surround the mRNA upstream of the E site codon were determined using mRNA analogues bearing the triplet UUU at the 3' end and the photoactivatable group on the guanosine or uridine residue, the modified nucleotide being located in position –4, –6 or –9.
MATERIALS AND METHODS
Materials
tRNAVal (1800 pmol/A260 unit) and tRNAPhe (1300 pmol/A260 unit) from E.coli were kindly provided by Dr T. Shapkina (B.P. Konstantinov's St Petersburg Institute of Nuclear Physics, Gatchina, Russia). DNA 20mers complementary to various sequences of human 18S rRNA were synthesized in the Institute of Chemical Biology and Fundamental Medicine.
mRNA analogues—derivatives of oligoribonucleotides (Table 1 and Figure 1)
Oligoribonucleotides were synthesized chemically using the solid-phase H-phosphonate method as described earlier (15).
Photoreactive derivatives of oligoribonucleotides bearing a perfluorophenylazide cross-linker at the N7 atom of the guanosine were obtained according to (11).
Oligoribonucleotide derivatives bearing a 4-azido-2,3,5,6-tetrafluorobenzoyl group attached to the C5 atom of the uridine residue via an ethylene diamine spacer were synthesized as described previously (16).
Prior to the cross-linking experiments, all photoreactive derivatives of oligoribonucleotides were labelled with 32P at the 5'-termini as described in (17).
Ribosomes, ribosomal complexes and cross-linking procedures
Ribosomes. 40S and 60S ribosomal subunits with intact rRNAs were isolated from unfrozen human placenta according to (18). Prior to use, the subunits were re-activated by incubating in binding buffer A (120 mM KCl, 13 mM MgCl2, 0.6 mM EDTA, 20 mM Tris–HCl, pH 7.5) at 37°C for 10 min. 80S ribosomes were obtained by association of the re-activated 40S and 60S subunits in a 40S:60S ratio of 1:1.3.
Ribosomal complexes were obtained by incubating 80S ribosomes (8 x 10–7 M) with mRNA analogues (5 x 10–6 M in the case of the hexamers and 2 x 10–6 M for nona- and dodecamers) and tRNA (4 x 10–6 M in the case of tRNAPhe and 7 x 10–6 M for tRNAVal) for 50 min at 20°C in buffer A. The extent of binding of the 5'-32P-labelled mRNAs to 80S ribosomes was examined by nitrocellulose filtration according to (19).
Irradiation of the complexes was carried out in ice-cooled cells during 0.5 min with UV light from a SpotCure (UVP) lamp via optical fibres. Shortwave UV light (wavelength < 280 nm) was cut-off by a thin glass filter. The irradiated complexes were completed with 1:30 (v/v) of 5% 2-mercaptoethanol and precipitated by 0.7 volume of ethanol at 0°C. The irradiated ribosomes were separated into subunits as described previously (8).
Analysis of distribution of the cross-linked labelled mRNA analogues between the 18S rRNA and proteins of the 40S subunit was carried out by PAGE in the presence of SDS as described in (8).
Analysis of the cross-linked ribosomal RNA, determination of regions of the 18S rRNA that contained the cross-linked mRNA analogues and of the cross-linked 18S rRNA nucleotides were carried out as described previously (20).
Analysis of the cross-linked 40S ribosomal proteins
Proteins were extracted from the solution of 40S subunits with acetic acid according to (21). Proteins cross-linked to mRNA analogues were identified by one-dimensional (1D) PAGE in the presence of SDS and by 2D-PAGE in two alternative systems as described (22). Separation in the first dimension was in an 8% gel containing 8 M urea at pH 8.6 in both systems; in the second dimension, separation was in a 12.5% gel containing SDS at pH 6.7 (system A) or in a 16% gel containing 8 M urea at pH 4.5 (system B). To identify the cross-linked proteins, the radioautograms were superimposed on the respective stained gels and the positions of the radioactive bands with respect to those of unmodified proteins were determined.
Cross-linking of mRNA analogue IV to proteins S10/S15
Total 40S proteins isolated from 200 pmol of 40S subunits were separated by 2D-PAGE in system A. A spot corresponding to proteins S10/S15 was cut out of the electrophoregram, stained with Coomassie R250, the proteins were eluted from the gel in buffer (20 mM Tris–HCl, pH 8.8 and 0.5% SDS) at 20°C for 24 h. The gel was removed by low speed centrifugation, to the supernatant was added sodium deoxycholate to 0.015%, the proteins were precipitated with 5% trichloroacetic acid, and the pellet was washed with mixture of ethanol and ether (1:1, v/v). The pellet was dissolved in 10 μl of 7 M urea containing 100 pmol of 5'-32P-labelled analogue IV, irradi-ated, supplied with 2-mercaptoethanol to a final concentration of 0.3% and precipitated as described above. To analyse the cross-linked proteins in system A, the pellet was dissolved in 4 μl of buffer (20 mM Tris-borate, pH 8.3, 1 mM EDTA, 8 M urea, 1% 2-mercaptoethanol and 0.1% pyranine G). To carry out the analysis in system B, the pellet was dissolved in the same volume of 8 M urea containing 1% 2-mercaptoethanol and pyranine G. Prior to electrophoresis, total 40S protein (isolated from 200 pmol of 40S subunits) dissolved in 1 μl of 8 M urea containing 1% 2-mercaptoethanol was added to the samples.
Identification of S26 protein by means of immunoblotting after the 1D SDS–PAGE separation of the proteins was performed according to (23) with specific rabbit antibodies obtained by affinity purification of antisera against human S26 (24).
RESULTS
mRNA analogues, model 80S ribosomal complexes and cross-linking
The mRNA analogues used in this study bore a perfluorophenylazide moiety at either the guanosine or the uridine residue (Table 1 and Figure 1). The analogues photocross-linked to human 80S ribosomes in three types of model complexes (Figure 1). In the first type, the cross-linker was at the first nucleotide of the P site bound codon (mRNA analogue I, position +1), in the second at any nucleotide of the A site bound codon (mRNA analogues II–VI, positions +4, +5 or +6), and in the third type, the modified nucleotide of the mRNA analogues was 5' of the P site bound codon (analogues VII–XII, positions –4, –6 and –9). The scheme for complex formation was based on the generally accepted idea that deacylated tRNA has a higher affinity for the ribosomal P site than for the A and E sites. Therefore, the location of mRNA I on the ribosome was governed by tRNAVal that directed codon GUU with the modified guanosine to the P site. mRNAs II–XII were positioned by tRNAPhe that directed codon UUU or UUC to the P site and therefore provided either the A site location of the adjacent modified codon (analogues II–VI), or the location of the modified nucleotide at position 5' of the E site bound codon (analogues VII–XII). Binary mixtures comprised of mRNA analogues with 80S ribosomes but without tRNA were used in control experiments. Typical binding levels for mRNA analogues were 0.70 and 0.15 mol per mol of 80S ribosomes in the ternary complexes obtained with tRNAPhe and tRNAVal, respectively. Without tRNA, the binding levels were from 0.03 to 0.05 mol per mol of ribosomes for the hexa- and heptamers and from 0.06 to 0.10 mol per mol of ribosomes for the nona- and dodecamers.
Table 1. mRNA analogues used for photocross-linking to human 80S ribosomes
Figure 1. Structures of modified nucleotides in mRNA analogues and the three types of 80S ribosomal complexes used here. The complexes with analogues I, III and VIII are presented as examples for each type of complex. The distance separating modified base and first nitrogen of the azidogroup is no more than 11 ? for U* and 14 ? for G* (13).
The cross-links were obtained by mild UV-irradiation of the preformed 80S ribosomal complexes. When irradiated ribosomes were separated into the 40S and 60S subunits, radioactive label (corresponding to the cross-linked mRNA analogue) was recovered mainly in the fractions containing the 40S subunits (data not shown), as in our previous studies with other mRNA analogues (9,10,12). Typical extents of cross-linking were 0.30 and 0.07 mol of mRNA per mol of 40S subunits for the ternary complexes with tRNAPhe and tRNAVal, respectively. In the binary mixtures, the cross-linking extent was from 0.02 to 0.03 mol of the mRNA per mol of 40S subunits. Analysis of the distribution of the cross-linked labelled mRNA analogues VII–XII between the 18S rRNA and proteins is presented in Figure 2: these mRNAs cross-linked only to ribosomal proteins since all the radioactive bands disappeared after treatment of the samples with proteinase K but remained after the treatment with RNase A (the latter treatment resulted in faster migration of the bands due to hydrolysis of the oligoribonuc-leotide cross-linked to the proteins). With the mRNA analogues I–VI, both rRNA and proteins were cross-linked (data not shown).
Figure 2. Autoradiogram of an SDS-PAGE analysis of 40S subunits isolated from the irradiated complexes of 80S ribosomes with analogues VII–IX (A) and X–XII (B). Lanes ‘+’ and ‘–’ correspond to the complexes obtained with and without tRNAPhe, respectively. Lanes PrK and RnA correspond to 40S subunits that were treated with proteinase K or RNase A prior to the analysis. Lane K (in B), 40S subunits isolated from the binary complex of 80S ribosomes with analogue XI formed in the presence of poly(U). The very left lanes marked Tp40 in each panel represent typical stained patterns; the positions of some ribosomal proteins are indicated according to (32,33).
Identification of 40S proteins cross-linked to analogues II–IV
At first, proteins isolated from the 40S subunits cross-linked to mRNA analogues II–IV were resolved by 1D-PAGE in the presence of SDS (Figure 3). Two radioactive bands corresponding to the proteins cross-linked to the labelled mRNA analogues are detected. The proteins corresponding to the faster migrating radioactive band are cross-linked only in the ternary complexes (Figure 3, lanes ‘+’), and efficiency of cross-linking evidently depends on the position of the cross-linker in the mRNA analogue. Radioactive bands of modified proteins are shifted towards the origin with respect to the stained bands of the corresponding unmodified proteins, since the cross-linked oligoribonucleotide retards electrophoretic migration of the modified protein. The value of the resulting shift in the 1D-PAGE is known from our previous studies (17,23,25). Taking this into account, candidates for the cross-linked proteins are S3, S4 and S6 for the upper band, and S10, S11, S13, S15, S17, S18, S24 and S25 for the lower band (Figure 3). One can note that with mRNA II the radioactive bands are located on the gel somewhat higher with respect to those with mRNAs III and IV. This is evidently due to the higher molecular mass of mRNA II (heptamer) with respect to the masses of mRNAs III and IV (hexamers).
Figure 3. Analysis of 40S ribosomal proteins cross-linked to 5'-32P-labelled mRNAs II–IV by 1D-PAGE in the presence of SDS. Lanes ‘–’ and ‘+’ correspond to the binary and ternary complexes, respectively. Lane K, typical stained gel, positions of the 40S ribosomal proteins are indicated according to (32,33).
Cross-linked proteins were identified more precisely by 2D-PAGE in two alternative systems. In system A separation was under alkaline conditions in the first dimension, and in the second dimension in a slightly acidic gel containing SDS. The value and direction of the shift that the cross-linked hexaribonucleotide imparts to the protein in this system is known from our previous studies (17,23,25). The band of the modified protein is shifted mainly in the first dimension (to the left with respect to the corresponding unmodified protein) where the negative charge of the cross-linked oligonucleotide significantly affects the mobility of the protein; the effect is stronger for proteins of low-molecular mass. The results of the analysis by 2D-PAGE of the proteins cross-linked to mRNA III in system A are presented in Figure 4. The patterns obtained with mRNAs II and IV are very similar (data not shown). Taking into account the data on the 1D-PAGE (see above), one can conclude that proteins S3 and S10/S15 cross-linked to mRNA analogues; modification of S10/S15 occurred only in the complexes formed with tRNAPhe. To clarify which protein S10 and/or S15 was modified, separation in system B (first dimension under alkaline conditions, and second at pH 4, both dimensions in the presence of urea) was carried out. Under these conditions, the cross-linked oligonucleotide retards the mobility of the protein in both dimensions, and the radioactive spot of the modified protein is shifted to left and above compared to the spot of the unmodified protein (17,23,25). The results of 2D-PAGE of the proteins cross-linked to mRNA III in system B are presented in Figure 5. Similar patterns were obtained for mRNAs II and IV (data not shown). The radioactive spot is shifted almost equally in both directions with respect to the spot of S14/S15, but is practically not shifted with respect to the spot of S10. Therefore, the cross-linked protein is S15 rather than S10. Interestingly, on the autoradiograms obtained in system B there is no radioactive spot that could be attributed to cross-linked protein S3 (Figure 5). Most probably, attachment of the hexanucleotide fragment to S3 deprives it of its ability to enter the gel in the first dimension as was detected earlier for protein S2 cross-linked to a hexaribonucleotide derivative (25).
Figure 4. Analysis of proteins cross-linked to mRNAs III in the ternary (A) and binary (B) complexes by 2D-PAGE in system A. Autoradiograms of the gels after separation of the proteins isolated from the modified 40S subunits. The cross-linked proteins are marked with an asterisk (*). (C) Typical stained gel, positions of the proteins are indicated according to (22). The locations of the radioactive spots corresponding to the cross-linked proteins are indicated by dotted lines.
Figure 5. Analysis of proteins cross-linked to mRNA III in the ternary complex by 2D-PAGE in system B. (A) Autoradiogram of the gel after separation of the proteins isolated from the modified 40S subunits. The cross-linked proteins are marked with an asterisk (*). (B) Typical stained gel, positions of the proteins are indicated according to (21). The location of the radioactive spot corresponding to the cross-linked protein is indicated by a dotted line.
To unambiguously confirm the modification of S15, analogue IV was cross-linked to the S10/S15 proteins directly (not within the 80S ribosomal complex). Since the identities of human 40S proteins in the 2D PAG electrophoregrams have been confirmed by N-terminal sequencing (21), proteins S10/S15 were isolated from the gel in which unmodified 40S proteins were resolved in system A (Figure 4C). To obtain the cross-link, the protein was mixed with mRNA analogue IV and irradiated. Analysis of the resulting cross-links of S10/S15 to mRNA IV by 2D-PAGE in systems A and B revealed radioactive spots at the same positions as in Figures 4 and 5 (data not shown). In addition, in system B an additional radioactive spot was seen at the very left boundary between the separating and spacer gels above the S10 protein spot; this radioactive spot evidently corresponded to the cross-linked S10 protein. Thus, the main protein cross-linked to mRNA analogues II–IV in the ternary 80S ribosomal complexes is indeed S15.
Identification of 40S proteins cross-linked to analogues VII–XII
Proteins cross-linked to analogues VII, IX, XI and XII were identified by 2D-PAGE in system A. Prior to analysis, the cross-linked oligoribonucleotide was hydrolysed with RNase A to decrease its effect on the electrophoretic mobilities of the proteins. This was possible for only those mRNA analogues in which the cross-linker was at the 5'-terminal nucleotide that bore 32P phosphate, and hydrolysis of such mRNA analogues did not lead to the loss of the label. The results of the 2D-PAGE analysis of proteins isolated from the 40S subunits cross-linked to mRNAs VIII and IX are presented in Figure 6. The patterns obtained for the ternary complexes with mRNA XI and XII (data not shown) were very similar to those with mRNAs VIII and IX, respectively. The patterns for the binary complexes were similar for all mRNA analogues. Figure 6 shows the pattern obtained with mRNA VIII as example. To identify the labelled proteins, we have taken into account the fact that the radioactive spots may be somewhat shifted to the left as compared with the corresponding unmodified proteins. This shift is caused by mono- or dinucleotide fragments that remain cross-linked to the protein after RNase A hydrolysis; these fragments bear two or three negative charges in basic media and thus somewhat retard the electrophoretic mobilities of the proteins in the first dimension. The data presented in Figure 6 for the ternary complexes (panels A and B) indicate that the main radioactive spot is adjacent to, and left of, the S26 spot in the stained gel. Since no other proteins except S26 are present in the gel to the right of the radioactive spot, one can conclude that protein S26 was the main target for cross-linking to the mRNA analogues. For the ternary complexes with analogues VIII and XI, one can also see a weak spot to the left of the S14 spot in the stained gel that most probably corresponds to minor cross-linking to protein S14 (Figure 6A). For the binary complexes, the only radioactive spot was left of the S3/S3a spot in the stained gel (Figure 6C). Cross-linking to protein S3a is unlikely since upon 1D separation, the upper radioactive band is lower than the band of unmodified S3a/S2 in the stained gel (Figure 2, lanes RnA ‘–’). Thus, protein S3 was cross-linked in the binary complexes. The same results were obtained for the complexes with analogues IX and XII (data not shown).
Figure 6. 2D-PAGE analysis of proteins cross-linked to mRNA analogues VIII (A and C) and IX (B) in system A. Autoradiograms of the gels after separation of the proteins isolated from the 40S subunits modified in the ternary (A,B) and binary (C) complexes; (D) typical stained gel, positions of the proteins are indicated according to (22). Positions of radioactive spots corresponding to the cross-linked proteins are marked with dotted lines.
Cross-linking of protein S26 to the mRNA in the ternary 80S ribosomal complexes was confirmed by immunoblotting; the data are presented in Figure 7 for analogues X–XII as examples. Antibodies raised against protein S26 produce an intense band corresponding to unmodified S26 (Figure 7B, all lanes) and a weaker band (lanes 1–3) whose position coincides with that of the intensive radioactive band in Figure 7A. The weaker band evidently corresponds to protein S26 cross-linked to the oligoribonucleotides. Protein S26 cross-linked to analogue XII has less electrophoretic mobility than that with analogues X and XI due to the larger molecular mass of the dodecamer XII as compared with the nonamers X and XI.
Figure 7. Identification of cross-linked S26 protein by immunoblotting after 1D-PAGE separation of 40S proteins with subsequent transfer onto a nitrocellulose membrane. Lanes 1, 2 and 3 correspond to the results with ternary complexes containing 80S ribosomes, tRNAPhe and analogue X, XI or XII, respectively. Lane K corresponds to a control experiment with proteins isolated from unmodified 40S subunits. (A) is an autoradiogram, (B) presents the identification of S26 with specific antibodies.
The results on cross-linking of mRNA analogues to ribosomal proteins are summarized in Table 2.
Table 2. Summary of the cross-linking data from positions –9 to +6 of mRNA on human 80S ribosomes obtained in this study
Identification of cross-linked nucleotides of 18S rRNA
RNA isolated from the irradiated ribosomal complexes was analysed by PAGE (data not shown). Cross-linking to the 18S rRNA was completely tRNA-dependent in all cases (no cross-linking occurs without tRNA). In ternary complexes, mRNA analogues cross-linked also to tRNA; this cross-linking was minor for the analogues II–VI and major for analogue I that modified tRNA more than 18S rRNA.
To determine the sequences of 18S rRNA containing cross-linked nucleotides, 18S rRNA isolated from irradiated complexes was digested with RNase H in the presence of deoxy-20mers complementary to various sequences of the 18S rRNA (from its 5'- to 3'-part) in parallel experiments. rRNA fragments resulting from hydrolysis were separated by denaturing PAGE, as in our previous studies (11,13,14). The results for all the mRNA analogues are summarized in Table 2.
The cross-linked nucleotides of 18S rRNA were identified by means of reverse transcription using primers chosen on the basis of the results of RNase H digestion (Figure 8). Primer extension leads to a stop or pause at the cross-linked rRNA nucleotide. The cross-linking site is generally assumed to be nucleotide 5' of the primer extension stop site. Therefore, the cross-linked 18S rRNA nucleotides were G626, G1702 and A1823–A1825. In particular, mRNA I cross-linked to nucleotide G1702 (Figure 8D), mRNAs II–VI to nucleotides A1823–A1825 (Figure 8A–C), and mRNAs V and VI also to G626 (Figure 8E). The stops are observed only for the respective ternary complexes of the ribosomes with the mRNA analogues and the cognate tRNA (lanes ‘+’). 18S rRNA samples modified with mRNA II and with mRNA I in the 80S ribosomal complex obtained with tRNAPhe were used here as controls since the rRNA nucleotides cross-linked with these mRNA analogues have been determined earlier (9,13). The results on the cross-linking of mRNA analogues to the 18S rRNA are summarized in Table 2.
Figure 8. Identification of sites of cross-linking of 18S rRNA to mRNA analogues by reverse transcription. Extension of 5'-32P-labelled primers complementary to 18S rRNA positions 1830–1849 (A–D) and 655–674 (E) on the 18S rRNA isolated from irradiated complexes. Lanes ‘–’ and ‘+’ correspond to the binary and ternary complexes, respectively. Lanes numbered ‘K’ are control 18S rRNA isolated from 80S ribosomes irradiated without mRNA analogues and treated in the same manner as the experimental samples. Lane marked ‘mRNA I*’ corresponds to 18S rRNA cross-linked with mRNA I in the 80S ribosomal complex obtained with tRNAPhe (guanosine with cross-linker at position +4). Lanes U, G, C, A, sequencing of 18S rRNA. Positions of the reverse transcription stops caused by the cross-linking are indicated.
DISCUSSION
mRNA analogues and model complexes
The design of the mRNA analogues was based on two main principles. First, the cross-linker was introduced at nucleotide positions that are not involved directly in Watson–Crick base pairing (C5 atoms of the uridines or N7 atoms of the guanos-ines). It had been previously shown that such modifications barely interfere with the coding properties of the triplets that contain the modified nucleotide (13). Second, binding of short mRNA analogues to 80S ribosomes is substantial only in the presence of a tRNA cognate to the selected mRNA codon that allows highly specific placement of the codon at the ribosomal P site. It should be noted that tRNAVal stimulated the binding of mRNA I much more weakly than the effect of tRNAPhe on the binding of mRNAs II–VI. This difference was caused by a higher affinity of tRNAPhe for its cognate codons UUU or UUC to the P site of the human 80S ribosome as compared with that of tRNAVal with its cognate codons (12). In general, the extent of cross-linking of mRNA analogue correlated with the level of its binding to the ribosomes.
Protein environment of mRNA on the ribosome
Positioning of mRNA with respect to 40S ribosomal proteins at the decoding site
In our earlier study (25), we reported that protein S15 cross-links to the derivative UUUGUU with a cross-linker on the guanosine residue when the latter is at position +1 or +4. Here, we have found this protein to cross-link with mRNA analogues bearing the cross-linker at the uridine residues if these are in positions from +4 to +6. For all these, the yield of the cross-link decreases significantly when the modified nucleotide moves from position +4 to position +6 (Figure 3). Therefore, we suggest that the first nucleotide of the A site codon is closest to the protein. Since nucleotides of the A site bound codon neighbour S15 and the 18S rRNA nucleotides A1823–A1825 and G626, it is reasonable to suggest that these rRNA nucleotides are in the vicinity of S15 at the decoding site of human 80S ribosomes. Cross-linking to S3 (together with other proteins) was detected previously when the modified uridine residues of the mRNA analogues, derivatives of UUUGUU, were at the P or E site (positions from –3 to +3) (10), and also for alkylating mRNA analogues when the modified nucleotide was at positions –6, –3, +6/+7 or +12/+13 (12). Protein S3 also cross-links to mRNA analogues without tRNA within labile binary complexes with 80S ribosomes, poorly detectable by the nitrocellulose filtration technique. This cross-linking actually occurs at the mRNA-binding site since it is inhibited by the addition of poly(U) (data not shown). Evidently, a site of tRNA-independent binding of mRNA analogues on the 80S ribosomes lies close to protein S3. Based on the data discussed one can suggest that in contrast to S15, protein S3 is close to the mRNA in various parts of the mRNA-binding channel of the 80S ribosomes, including its regions distant from the decoding site.
Arrangement of the upstream mRNA region
Modified mRNA nucleotides cross-link only to 40S ribosomal proteins from positions –4 to –9. The cross-linking patterns are almost insensitive to the nature of the nucleotide and the position of the nucleotide regarding the P site bound codon within the interval –4 to –9. This indicates that the upstream mRNA region is surrounded mainly by proteins on the 80S ribosome. The sets of proteins cross-linked to the modified uridines and guanosines from positions –4 to –9 are very similar, and the main target for cross-linking in all cases is S26. The same protein was earlier found to cross-link from mRNA positions +1 and –3 (10); thus, S26 is close to at least 10 mRNA nucleotides at positions from –9 to +1. Earlier, using alkylating mRNA analogues it was reported that the reactive group in the 5'-terminal nucleotide at positions from –6 to +12/13 cross-linked to proteins S3 and S3a (20). The difference is evidently due to the different natures of the alkylating and photoreactive cross-linkers in the mRNA analogues that may result in the formation of different modification products .
In addition, minor cross-linking to protein S14 from position –6 (see the data for analogues VIII and XI in Figures 2 and 6) points to the possible proximity between this protein and the mRNA at nucleotide position –6. It is worthy of notice that it was proposed on the basis of the cryoelectron microscopy data of mammalian ribosomes that S14 is located close to the mRNA exit tunnel (26) where the 5'-terminal sequence of the ribosome-bound portion of the mRNA is positioned. In binary complexes with 80S ribosomes, mRNA analogues VII–XII cross-link to protein S3 as do analogues II–VI (see above). Clearly, without tRNA short mRNA analogues bind to 80S ribosomes at a site close to protein S3.
Positioning of mRNA with respect to 18S rRNA at the decoding site
The +1/ G1702 cross-link was found for the first time in this study. This cross-link is relatively weak since tRNAVal bound at the P site (Figure 1, complex 1) shields 18S rRNA from cross-linking. This assumption is confirmed by the data on cross-linking of mRNA I to 18S rRNA in 80S ribosomal complexes where the modified GUU codon was bound to the A site and the cross-linker was in position +4 (13). In this case, the binding of a tRNA molecule in the A site resulted in significant decrease of cross-linking of the mRNA analogue to nucleotides A1823 and A1824 of the 18S rRNA.
Earlier it was shown that mRNA analogues with a perfluoro-phenylazide group at either the 5'-phosphate or the C5 atom of the 5'-terminal uridine at position +1 cross-linked to nucleotide G1207 of the 18S rRNA (13,14). In the present study, the same cross-linker at the N7 atom of the guanosine in the middle part of the mRNA analogue modified G1702, and no cross-linking was found in other regions of the 18S rRNA. These differences seem to be caused by different spatial orientations of the cross-linkers. Cross-linking of different modified mRNA nucleotides from position +1 to nucleotides G1207 (in the middle of helix 28) and G1702 (at the beginning of helix 44) is not surprising since these nucleotides are located close to each other in the secondary structure of the 18S rRNA (see www.rna.utexas.edu). It was previously reported that nucleotide G1702 cross-linked to the 3'-terminal alkylating group of mRNA analogues when the corresponding 3'-terminal nucleotide was at positions +3/+4 and +6/+7 (12). Cross-linking from positions +1, +3/+4 and +6/+7 to the same nucleotide G1702 of 18S rRNA may reflect the existence of a turn between codons bound at the A and P sites of the 80S ribosome.
In our previous reports (9,13) using mRNAs I and II, we showed that 18S rRNA nucleotides A1823 and A1824 are close to the first nucleotide of the codon bound to the A site of human ribosomes. The results of the present study for the first time show that all nucleotides of the A site bound codon are in the vicinity of nucleotides A1823–A1825 of the 18S rRNA. Moreover, we found that the second and third nucleotides of this codon are also close to G626. The latter was cross-linked only in the case of the guanosine-modified mRNA analogues V and VI. The lack of cross-linking of G626 with the modified uridine residues at the same positions (mRNAs III and IV) may be explained by different geometries of the spacers that link the photoactivatable moieties to the uridine and the guanosine residues (Figure 1). A cross-linker at the uridine may be unable to reach G626.
Extent of conservation of the mRNA-binding track on the ribosome
Location of template on the human ribosome
The results of this study together with previous cross-linking data on human ribosomes (9–11,13,20) provide a view on the positioning of the mRNA on the human ribosome. According to the scheme presented in Figure 9, nucleotide G961 of the 18S rRNA is in the vicinity of mRNA positions –3 and –2; nucleotide G1207 is close to mRNA positions from –3 to +3, nucleotide G1702 is close to the P site bound codon (position +1 to +3), nucleotides A1823–A1825 are in the proximity of the A site bound codon (positions +4 to +6), and nucleotide G626 is close to two nucleotides of the A site bound codon (positions +5 and +6). Protein S26 surrounds the mRNA positions from –9 to +1, whereas S15 is in the vicinity of P and A site bound codons (positions +1 to +6). Protein S3a most probably neighbours the long mRNA region including a portion the upstream region, the region of the codon–anticodon interactions and a portion of the downstream region.
Figure 9. Scheme of the mutual location of a template and the structural elements of the 40S subunit in the region of the mRNA-binding site of human ribosomes. Large circles, ribosomal proteins; small circles, nucleotides of 18S rRNA.
Variability of protein environment of mRNA on the ribosome
Cryo-electron microscopy data on eukaryotic ribosomes show that the structures of the active sites of pro- and eukaryotic ribosomes are in general similar (26,27). Nevertheless, a comparison of the tRNA-dependent cross-links of human 80S ribosomes with the X-ray crystallographic data obtained with prokaryotic ribosomes (1–4) does not reveal visible similarities in the proteins neighbouring the A site bound codon between prokaryotic and mammalian ribosomes. In the prokaryotic ribosome, only protein S12 is located close to the decoding site. Eukaryotic protein S23, the homologue of the prokaryotic S12 protein (28), did not cross-link to any mRNA analogue in the present study. According to our cross-linking data, all the nucleotides of the A site codon lie close to protein S15 on the 80S ribosome. Its prokaryotic homologue S19 (28), located mainly on the head of the 30S subunit away from the mRNA track has a tail that extends towards the decoding site (1,4). However, the minimum distance between the tail and the A site bound codon calculated from models (1,4) is between 39 ? (position +4) and 45 ? (position +6). If the spatial structure of mammalian S15 in the 40S subunit is similar to that of S19 in the 30S subunit (i.e. minimum distance between the tail of S15 and the A site bound codon is 40 ?), a cross-linker with a length of no more than 11 ? attached to nucleotides of the A site bound codon could not reach the tail of S15. The position of the main globular part of S15 on the head of the 40S subunit (27) is similar to that of S19 on the head of the 30S subunit (29). Based on the results of this study, we suggest that the tail of mammalian S15 in the 40S subunit comes substantially closer to the decoding site than the tail of prokaryotic S19 in the 30S subunit.
Other more distinct differences concern the environment of the mRNA region upstream of the E site bound codon (positions –9 to –4). First, mammalian proteins S26 (this study) and S3a (12,20) that have no homologues among prokaryotic ribosomal proteins (28) predominantly surround this mRNA region . Second, the 18S rRNA was not found in the molecular environment of the upstream part of the mRNA. In contrast, according to X-ray crystallographic data on prokaryotic 70S ribosomes (4), the immediate molecular environment of mRNA positions –9 to –4 contains mainly 16S rRNA, and protein S11 interacts with the backbone of the mRNA around positions –6 to –4. Data on the spatial structures of eukaryotic proteins S3a and S26 and the location of these proteins on the 40S subunit are not available; therefore it is difficult to conclude which parts of the proteins lie close to the mRNA upstream of the E site bound codon. However, it seems more probable that the upstream part of the mRNA is close to the main part of S26 since a long stretch of mRNA from positions –9 to +1 was found to neighbour this protein. One can suggest that S26 shields 18S rRNA and protein S14 from contacts with the upstream part of the mRNA.
Conservation of positioning of mRNA at the decoding site of the small subunit rRNA
Nucleotides A1824, A1825, G1702 and G626 of the 18S rRNA are conserved in the secondary structure of the small rRNA subunit and correspond to A1492, A1493, G1401 and G530 of the prokaryotic 16S rRNA (see www.rna.utexas.edu and Figure 10). It is known that nucleotides A1492 and A1493 of 16S rRNA are involved in the decoding of prokaryotic ribosomes and play a key role in the discrimination of correct codon–anticodon duplex at the A site (31). According to X-ray crystallographic data (1,3,4), nucleotides A1492, A1493, G1401 and G530 of 16S rRNA participate directly in the accommodation of mRNA codons in the region of codon–anticodon interactions on prokaryotic ribosomes. Thus, the phosphate of G1401 is located between the third nucleotide of the P site codon and the first nucleotide of the A site codon and produces a kink in the mRNA between the A and P site codons. Nucleotides A1492, A1493 and G530 are involved in the formation of the decoding site. A1493 facilitates fixation of the first pair of the codon–anticodon duplex at the A site by hydrogen bonds between the first nucleotide of the codon and the last nucleotide of the anticodon. A1492 interacts with the second nucleotide of the A site codon, and G530 interacts with the second nucleotide of the anticodon; G530 also takes part in the fixation of third pair in the codon–anticodon duplex (3,4). The results on human ribosomes clearly are in remarkable agreement with X-ray crystallographic data on prokaryotic ribosomes. Consequently, the data obtained reveal the highly conserved nature of the decoding site of the small subunit rRNA for all classes of ribosomes.
Figure 10. Fragment of 18S rRNA secondary structure (from www.rna.utexas.edu) containing conserved nucleotides capable of cross-linking to various positions of mRNA analogues (indicated in boxes). The corresponding nucleotides of E.coli 16S rRNA are numbered in brackets.
Concluding remarks
Our study experimentally confirms the widely accepted idea on the conserved rRNA ‘core’ of the ribosome that appears to be structurally similar in all kingdoms. At the same time, our findings indicate noticeable dissimilarities in the molecular environment of the mRNA in pro- and eukaryotic ribosomes. The first concerns proteins neighbouring the decoding site and seems to be caused by different positioning of the tails of mammalian S15 and its prokaryotic homologue S19 with respect to this site. Other more important differences are related to the molecular environment of the mRNA region upstream of the E site bound codon which on 80S ribosomes is surrounded largely by proteins, in contrast to 70S ribosomes in which the environment of this part of the mRNA consists of both rRNA and proteins. The proteins surrounding the upstream part of the mRNA on the 80S ribosome have no prokaryotic homologues. The latter differences may be related to the more complicated process of translation regulation in eukaryotes as compared with that in prokaryotes. Probably, the binding site of the upstream part of the mRNA on the 80S ribosome is formed by eukaryotic-specific proteins that may be targets for binding of various regulatory factors (such as chaperones, modifying enzymes, etc.) capable of affecting the accuracy and rate of translation. Future structural and biochemical experiments will be required to address these aspects of the complicated mechanisms used by the eukaryotic ribosomal machinery.
ACKNOWLEDGEMENTS
We gratefully thank Anne-Lise Haenni and Richard Brimacombe for critical reading of this manuscript. This work was supported by the Russian Foundation for Basic Research (grant no. 02-04-48194 to G.K.).
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* To whom correspondence should be addressed. Tel: +3832 35 62 29; Fax: +3832 33 36 77; Email: karpova@niboch.nsc.ru
ABSTRACT
This study is centred upon an important biological problem concerning the structural organization of mammalian ribosomes that cannot be studied by X-ray analysis because 80S ribosome crystals are still unavailable. Here, positioning of the mRNA on 80S ribosomes was studied using mRNA analogues containing the perfluorophenylazide cross-linker on either the guanosine or an uridine residue. The modi-fied nucleotides were directed to positions from –9 to +6 with respect to the first nucleotide of the P site bound codon by a tRNA cognate to the triplet targeted to the P site. Upon mild UV-irradiation, the modified nucleotides at positions +4 to +6 cross-linked to protein S15 and 18S rRNA nucleotides A1823–A1825. In addition, modified guanosines in positions +5 and +6 also cross-linked to G626, and in position +1 to G1702. Cross-linking from the upstream positions was mainly to protein S26 that has no prokaryotic homologues. These findings indicate that the tail of mammalian S15 comes closer to the decoding site than that of its prokaryotic homologue S19, and that the environments of the upstream part of mRNA on 80S and 70S ribosomes differ. On the other hand, the results confirm the widely accepted idea regarding the conserved nature of the decoding site of the small subunit rRNA.
INTRODUCTION
Genetic information is transmitted to the ribosome where trinucleotide codons of the mRNA are recognized by anticodons of the cognate tRNAs at the decoding site. This function is common to prokaryotic and eukaryotic ribosomes. The structure of the decoding site of the prokaryotic ribosome and the molecular basis for decoding are known today at the atomic level thanks to the remarkable progress of X-ray crystallographic analysis (1–4). It is generally assumed that arrangement of the mRNA on the eukaryotic ribosome resembles that on the prokaryotic ribosome. Nevertheless, little is known about the structure of the mRNA-binding site of eukaryotic ribosomes, because many methods used to study prokaryotic ribosomes are not applicable to eukaryotic ribosomes yet. For instance, still crystals of eukaryotic ribosomal subunits suitable for X-ray analysis are not available. Approaches based on reconstitution of functionally active eukaryotic ribosomal subunits from ribosomal proteins and rRNAs also are not applicable to study eukaryotic ribosomes since to date no methodology is available for the reconstitution of the eukaryotic ribosomal subunits in vitro. One approach that can provide detailed information on the structures of functional sites of eukaryotic ribosomes is site-directed cross-linking. The method is based on the use of mRNA analogues that contain chemically reactive groups at defined positions . This makes it possible to determine the ribosomal components contacting—or located close to—the modified mRNA nucleotides. The majority of the data on positioning of the mRNA on the prokaryotic ribosome obtained by means of the site-directed cross-linking approach turned out to be in general agreement with recent X-ray crystallographic data (1–4). This justifies the use of cross-linking approach to study functional sites of eukaryotic ribosomes.
In earlier studies using s4U-containing mRNA analogues, the substantial differences in arrangement of mRNA on human and Escherichia coli ribosomes have been found. In particular, these mRNA analogues produced much fewer numbers of cross-links with 18S rRNA in the 80S ribosome than with 16S rRNA in the 70S ribosome (6,7). It was suggested that the mRNA track is stricter in mammalian ribosomes than in eubacterial ribosomes, or that internal flexibility of mammalian ribosomes is lower than that of eubacterial ribosomes (7). A very limited number of cross-links of s4U-containing mRNA analogues to 18S rRNA in human ribosomes might also be due to other reasons. Cross-links would not occur if the mRNA nucleotide contacts the 18S rRNA by its ribose-phosphate backbone, or if the contacting mRNA and rRNA bases were in mutual orientations not suitable for the cross-linking. In addition, large portions of the mRNA on the human ribosome might be in contact with proteins rather than with the rRNA. In any event, these data indicate that the mRNA-binding track is arranged differently on prokaryotic than on eukaryotic ribosomes. These differences can be studied more extensively by means of mRNA analogues with conformationally flexible cross-linkers capable of reaching any ribosomal component in the vicinity of the modified mRNA nucleotide.
Short mRNA analogues, derivatives of oligoribonucleotides (from hexa- to dodecamers) that contain a perfluoroarylazide cross-linker at different locations, turned out to be suitable tools for studing mRNA-binding site of eukaryotic (in particular, human) ribosomes (8–12). These templates may be positioned exactly on the 80S ribosome by the addition of tRNA cognate to the selected mRNA codon targeted to the P site, since short oligoribonucleotides barely bind to the ribosomes in the absence of tRNA (9,10,12). Positioning of short mRNA analogues on the 80S ribosome is functionally correct because the ribosome is capable of translating such analogues to form oligopeptides encoded by the mRNA sequences (12). Perfluorophenylazide groups attached to the selected nucleotides of the mRNA analogue via conformationally flexible spacers are appropriate cross-linkers capable of effective attachment to rRNA as well as to proteins in the ribosome (12). Using a set of short mRNA analogues bearing the cross-linker at either the N7 atom of the guanosine or at the 5'-terminal nucleotide (either to the 5'-phosphate or C5 atom of uridine), we have determined 18S rRNA nucleotides and ribosomal proteins neighbouring the P and E site bound codons and the first nucleotide of the A site bound codon on human 80S ribosome (10,11,13). For the cross-linker at mRNA positions +1 and –3 with respect to the first nucleotide of the P site bound codon, the target for modification in the 80S ribosome was sensitive to the nature of the nucleotide that bore the cross-linker (11,13,14). Evidently, the application of a set of mRNA analogues in which the nature of the nucleotides carrying the photoactivatable groups varies, provides more comprehensive information on the environment of mRNA nucleotides on the ribosome.
In the present study, to determine the 18S rRNA nucleotides neighbouring first nucleotide of the P site bound codon, a derivative of the hexaribonucleotide UUUGUU (containing the triplets UUU and GUU coding for Phe and Val, respectively) with a perfluorophenylazide group at the N7 atom of the guanosine was used. To reveal 18S rRNA nucleotides and ribosomal proteins in the vicinity of the A site bound codon, a set of oligomers (from hexa- to nonamers) with a perfluorophenylazide group at either the N7 atom of the guanosine or the C5 atom of the uridine was used. Ribosomal components that surround the mRNA upstream of the E site codon were determined using mRNA analogues bearing the triplet UUU at the 3' end and the photoactivatable group on the guanosine or uridine residue, the modified nucleotide being located in position –4, –6 or –9.
MATERIALS AND METHODS
Materials
tRNAVal (1800 pmol/A260 unit) and tRNAPhe (1300 pmol/A260 unit) from E.coli were kindly provided by Dr T. Shapkina (B.P. Konstantinov's St Petersburg Institute of Nuclear Physics, Gatchina, Russia). DNA 20mers complementary to various sequences of human 18S rRNA were synthesized in the Institute of Chemical Biology and Fundamental Medicine.
mRNA analogues—derivatives of oligoribonucleotides (Table 1 and Figure 1)
Oligoribonucleotides were synthesized chemically using the solid-phase H-phosphonate method as described earlier (15).
Photoreactive derivatives of oligoribonucleotides bearing a perfluorophenylazide cross-linker at the N7 atom of the guanosine were obtained according to (11).
Oligoribonucleotide derivatives bearing a 4-azido-2,3,5,6-tetrafluorobenzoyl group attached to the C5 atom of the uridine residue via an ethylene diamine spacer were synthesized as described previously (16).
Prior to the cross-linking experiments, all photoreactive derivatives of oligoribonucleotides were labelled with 32P at the 5'-termini as described in (17).
Ribosomes, ribosomal complexes and cross-linking procedures
Ribosomes. 40S and 60S ribosomal subunits with intact rRNAs were isolated from unfrozen human placenta according to (18). Prior to use, the subunits were re-activated by incubating in binding buffer A (120 mM KCl, 13 mM MgCl2, 0.6 mM EDTA, 20 mM Tris–HCl, pH 7.5) at 37°C for 10 min. 80S ribosomes were obtained by association of the re-activated 40S and 60S subunits in a 40S:60S ratio of 1:1.3.
Ribosomal complexes were obtained by incubating 80S ribosomes (8 x 10–7 M) with mRNA analogues (5 x 10–6 M in the case of the hexamers and 2 x 10–6 M for nona- and dodecamers) and tRNA (4 x 10–6 M in the case of tRNAPhe and 7 x 10–6 M for tRNAVal) for 50 min at 20°C in buffer A. The extent of binding of the 5'-32P-labelled mRNAs to 80S ribosomes was examined by nitrocellulose filtration according to (19).
Irradiation of the complexes was carried out in ice-cooled cells during 0.5 min with UV light from a SpotCure (UVP) lamp via optical fibres. Shortwave UV light (wavelength < 280 nm) was cut-off by a thin glass filter. The irradiated complexes were completed with 1:30 (v/v) of 5% 2-mercaptoethanol and precipitated by 0.7 volume of ethanol at 0°C. The irradiated ribosomes were separated into subunits as described previously (8).
Analysis of distribution of the cross-linked labelled mRNA analogues between the 18S rRNA and proteins of the 40S subunit was carried out by PAGE in the presence of SDS as described in (8).
Analysis of the cross-linked ribosomal RNA, determination of regions of the 18S rRNA that contained the cross-linked mRNA analogues and of the cross-linked 18S rRNA nucleotides were carried out as described previously (20).
Analysis of the cross-linked 40S ribosomal proteins
Proteins were extracted from the solution of 40S subunits with acetic acid according to (21). Proteins cross-linked to mRNA analogues were identified by one-dimensional (1D) PAGE in the presence of SDS and by 2D-PAGE in two alternative systems as described (22). Separation in the first dimension was in an 8% gel containing 8 M urea at pH 8.6 in both systems; in the second dimension, separation was in a 12.5% gel containing SDS at pH 6.7 (system A) or in a 16% gel containing 8 M urea at pH 4.5 (system B). To identify the cross-linked proteins, the radioautograms were superimposed on the respective stained gels and the positions of the radioactive bands with respect to those of unmodified proteins were determined.
Cross-linking of mRNA analogue IV to proteins S10/S15
Total 40S proteins isolated from 200 pmol of 40S subunits were separated by 2D-PAGE in system A. A spot corresponding to proteins S10/S15 was cut out of the electrophoregram, stained with Coomassie R250, the proteins were eluted from the gel in buffer (20 mM Tris–HCl, pH 8.8 and 0.5% SDS) at 20°C for 24 h. The gel was removed by low speed centrifugation, to the supernatant was added sodium deoxycholate to 0.015%, the proteins were precipitated with 5% trichloroacetic acid, and the pellet was washed with mixture of ethanol and ether (1:1, v/v). The pellet was dissolved in 10 μl of 7 M urea containing 100 pmol of 5'-32P-labelled analogue IV, irradi-ated, supplied with 2-mercaptoethanol to a final concentration of 0.3% and precipitated as described above. To analyse the cross-linked proteins in system A, the pellet was dissolved in 4 μl of buffer (20 mM Tris-borate, pH 8.3, 1 mM EDTA, 8 M urea, 1% 2-mercaptoethanol and 0.1% pyranine G). To carry out the analysis in system B, the pellet was dissolved in the same volume of 8 M urea containing 1% 2-mercaptoethanol and pyranine G. Prior to electrophoresis, total 40S protein (isolated from 200 pmol of 40S subunits) dissolved in 1 μl of 8 M urea containing 1% 2-mercaptoethanol was added to the samples.
Identification of S26 protein by means of immunoblotting after the 1D SDS–PAGE separation of the proteins was performed according to (23) with specific rabbit antibodies obtained by affinity purification of antisera against human S26 (24).
RESULTS
mRNA analogues, model 80S ribosomal complexes and cross-linking
The mRNA analogues used in this study bore a perfluorophenylazide moiety at either the guanosine or the uridine residue (Table 1 and Figure 1). The analogues photocross-linked to human 80S ribosomes in three types of model complexes (Figure 1). In the first type, the cross-linker was at the first nucleotide of the P site bound codon (mRNA analogue I, position +1), in the second at any nucleotide of the A site bound codon (mRNA analogues II–VI, positions +4, +5 or +6), and in the third type, the modified nucleotide of the mRNA analogues was 5' of the P site bound codon (analogues VII–XII, positions –4, –6 and –9). The scheme for complex formation was based on the generally accepted idea that deacylated tRNA has a higher affinity for the ribosomal P site than for the A and E sites. Therefore, the location of mRNA I on the ribosome was governed by tRNAVal that directed codon GUU with the modified guanosine to the P site. mRNAs II–XII were positioned by tRNAPhe that directed codon UUU or UUC to the P site and therefore provided either the A site location of the adjacent modified codon (analogues II–VI), or the location of the modified nucleotide at position 5' of the E site bound codon (analogues VII–XII). Binary mixtures comprised of mRNA analogues with 80S ribosomes but without tRNA were used in control experiments. Typical binding levels for mRNA analogues were 0.70 and 0.15 mol per mol of 80S ribosomes in the ternary complexes obtained with tRNAPhe and tRNAVal, respectively. Without tRNA, the binding levels were from 0.03 to 0.05 mol per mol of ribosomes for the hexa- and heptamers and from 0.06 to 0.10 mol per mol of ribosomes for the nona- and dodecamers.
Table 1. mRNA analogues used for photocross-linking to human 80S ribosomes
Figure 1. Structures of modified nucleotides in mRNA analogues and the three types of 80S ribosomal complexes used here. The complexes with analogues I, III and VIII are presented as examples for each type of complex. The distance separating modified base and first nitrogen of the azidogroup is no more than 11 ? for U* and 14 ? for G* (13).
The cross-links were obtained by mild UV-irradiation of the preformed 80S ribosomal complexes. When irradiated ribosomes were separated into the 40S and 60S subunits, radioactive label (corresponding to the cross-linked mRNA analogue) was recovered mainly in the fractions containing the 40S subunits (data not shown), as in our previous studies with other mRNA analogues (9,10,12). Typical extents of cross-linking were 0.30 and 0.07 mol of mRNA per mol of 40S subunits for the ternary complexes with tRNAPhe and tRNAVal, respectively. In the binary mixtures, the cross-linking extent was from 0.02 to 0.03 mol of the mRNA per mol of 40S subunits. Analysis of the distribution of the cross-linked labelled mRNA analogues VII–XII between the 18S rRNA and proteins is presented in Figure 2: these mRNAs cross-linked only to ribosomal proteins since all the radioactive bands disappeared after treatment of the samples with proteinase K but remained after the treatment with RNase A (the latter treatment resulted in faster migration of the bands due to hydrolysis of the oligoribonuc-leotide cross-linked to the proteins). With the mRNA analogues I–VI, both rRNA and proteins were cross-linked (data not shown).
Figure 2. Autoradiogram of an SDS-PAGE analysis of 40S subunits isolated from the irradiated complexes of 80S ribosomes with analogues VII–IX (A) and X–XII (B). Lanes ‘+’ and ‘–’ correspond to the complexes obtained with and without tRNAPhe, respectively. Lanes PrK and RnA correspond to 40S subunits that were treated with proteinase K or RNase A prior to the analysis. Lane K (in B), 40S subunits isolated from the binary complex of 80S ribosomes with analogue XI formed in the presence of poly(U). The very left lanes marked Tp40 in each panel represent typical stained patterns; the positions of some ribosomal proteins are indicated according to (32,33).
Identification of 40S proteins cross-linked to analogues II–IV
At first, proteins isolated from the 40S subunits cross-linked to mRNA analogues II–IV were resolved by 1D-PAGE in the presence of SDS (Figure 3). Two radioactive bands corresponding to the proteins cross-linked to the labelled mRNA analogues are detected. The proteins corresponding to the faster migrating radioactive band are cross-linked only in the ternary complexes (Figure 3, lanes ‘+’), and efficiency of cross-linking evidently depends on the position of the cross-linker in the mRNA analogue. Radioactive bands of modified proteins are shifted towards the origin with respect to the stained bands of the corresponding unmodified proteins, since the cross-linked oligoribonucleotide retards electrophoretic migration of the modified protein. The value of the resulting shift in the 1D-PAGE is known from our previous studies (17,23,25). Taking this into account, candidates for the cross-linked proteins are S3, S4 and S6 for the upper band, and S10, S11, S13, S15, S17, S18, S24 and S25 for the lower band (Figure 3). One can note that with mRNA II the radioactive bands are located on the gel somewhat higher with respect to those with mRNAs III and IV. This is evidently due to the higher molecular mass of mRNA II (heptamer) with respect to the masses of mRNAs III and IV (hexamers).
Figure 3. Analysis of 40S ribosomal proteins cross-linked to 5'-32P-labelled mRNAs II–IV by 1D-PAGE in the presence of SDS. Lanes ‘–’ and ‘+’ correspond to the binary and ternary complexes, respectively. Lane K, typical stained gel, positions of the 40S ribosomal proteins are indicated according to (32,33).
Cross-linked proteins were identified more precisely by 2D-PAGE in two alternative systems. In system A separation was under alkaline conditions in the first dimension, and in the second dimension in a slightly acidic gel containing SDS. The value and direction of the shift that the cross-linked hexaribonucleotide imparts to the protein in this system is known from our previous studies (17,23,25). The band of the modified protein is shifted mainly in the first dimension (to the left with respect to the corresponding unmodified protein) where the negative charge of the cross-linked oligonucleotide significantly affects the mobility of the protein; the effect is stronger for proteins of low-molecular mass. The results of the analysis by 2D-PAGE of the proteins cross-linked to mRNA III in system A are presented in Figure 4. The patterns obtained with mRNAs II and IV are very similar (data not shown). Taking into account the data on the 1D-PAGE (see above), one can conclude that proteins S3 and S10/S15 cross-linked to mRNA analogues; modification of S10/S15 occurred only in the complexes formed with tRNAPhe. To clarify which protein S10 and/or S15 was modified, separation in system B (first dimension under alkaline conditions, and second at pH 4, both dimensions in the presence of urea) was carried out. Under these conditions, the cross-linked oligonucleotide retards the mobility of the protein in both dimensions, and the radioactive spot of the modified protein is shifted to left and above compared to the spot of the unmodified protein (17,23,25). The results of 2D-PAGE of the proteins cross-linked to mRNA III in system B are presented in Figure 5. Similar patterns were obtained for mRNAs II and IV (data not shown). The radioactive spot is shifted almost equally in both directions with respect to the spot of S14/S15, but is practically not shifted with respect to the spot of S10. Therefore, the cross-linked protein is S15 rather than S10. Interestingly, on the autoradiograms obtained in system B there is no radioactive spot that could be attributed to cross-linked protein S3 (Figure 5). Most probably, attachment of the hexanucleotide fragment to S3 deprives it of its ability to enter the gel in the first dimension as was detected earlier for protein S2 cross-linked to a hexaribonucleotide derivative (25).
Figure 4. Analysis of proteins cross-linked to mRNAs III in the ternary (A) and binary (B) complexes by 2D-PAGE in system A. Autoradiograms of the gels after separation of the proteins isolated from the modified 40S subunits. The cross-linked proteins are marked with an asterisk (*). (C) Typical stained gel, positions of the proteins are indicated according to (22). The locations of the radioactive spots corresponding to the cross-linked proteins are indicated by dotted lines.
Figure 5. Analysis of proteins cross-linked to mRNA III in the ternary complex by 2D-PAGE in system B. (A) Autoradiogram of the gel after separation of the proteins isolated from the modified 40S subunits. The cross-linked proteins are marked with an asterisk (*). (B) Typical stained gel, positions of the proteins are indicated according to (21). The location of the radioactive spot corresponding to the cross-linked protein is indicated by a dotted line.
To unambiguously confirm the modification of S15, analogue IV was cross-linked to the S10/S15 proteins directly (not within the 80S ribosomal complex). Since the identities of human 40S proteins in the 2D PAG electrophoregrams have been confirmed by N-terminal sequencing (21), proteins S10/S15 were isolated from the gel in which unmodified 40S proteins were resolved in system A (Figure 4C). To obtain the cross-link, the protein was mixed with mRNA analogue IV and irradiated. Analysis of the resulting cross-links of S10/S15 to mRNA IV by 2D-PAGE in systems A and B revealed radioactive spots at the same positions as in Figures 4 and 5 (data not shown). In addition, in system B an additional radioactive spot was seen at the very left boundary between the separating and spacer gels above the S10 protein spot; this radioactive spot evidently corresponded to the cross-linked S10 protein. Thus, the main protein cross-linked to mRNA analogues II–IV in the ternary 80S ribosomal complexes is indeed S15.
Identification of 40S proteins cross-linked to analogues VII–XII
Proteins cross-linked to analogues VII, IX, XI and XII were identified by 2D-PAGE in system A. Prior to analysis, the cross-linked oligoribonucleotide was hydrolysed with RNase A to decrease its effect on the electrophoretic mobilities of the proteins. This was possible for only those mRNA analogues in which the cross-linker was at the 5'-terminal nucleotide that bore 32P phosphate, and hydrolysis of such mRNA analogues did not lead to the loss of the label. The results of the 2D-PAGE analysis of proteins isolated from the 40S subunits cross-linked to mRNAs VIII and IX are presented in Figure 6. The patterns obtained for the ternary complexes with mRNA XI and XII (data not shown) were very similar to those with mRNAs VIII and IX, respectively. The patterns for the binary complexes were similar for all mRNA analogues. Figure 6 shows the pattern obtained with mRNA VIII as example. To identify the labelled proteins, we have taken into account the fact that the radioactive spots may be somewhat shifted to the left as compared with the corresponding unmodified proteins. This shift is caused by mono- or dinucleotide fragments that remain cross-linked to the protein after RNase A hydrolysis; these fragments bear two or three negative charges in basic media and thus somewhat retard the electrophoretic mobilities of the proteins in the first dimension. The data presented in Figure 6 for the ternary complexes (panels A and B) indicate that the main radioactive spot is adjacent to, and left of, the S26 spot in the stained gel. Since no other proteins except S26 are present in the gel to the right of the radioactive spot, one can conclude that protein S26 was the main target for cross-linking to the mRNA analogues. For the ternary complexes with analogues VIII and XI, one can also see a weak spot to the left of the S14 spot in the stained gel that most probably corresponds to minor cross-linking to protein S14 (Figure 6A). For the binary complexes, the only radioactive spot was left of the S3/S3a spot in the stained gel (Figure 6C). Cross-linking to protein S3a is unlikely since upon 1D separation, the upper radioactive band is lower than the band of unmodified S3a/S2 in the stained gel (Figure 2, lanes RnA ‘–’). Thus, protein S3 was cross-linked in the binary complexes. The same results were obtained for the complexes with analogues IX and XII (data not shown).
Figure 6. 2D-PAGE analysis of proteins cross-linked to mRNA analogues VIII (A and C) and IX (B) in system A. Autoradiograms of the gels after separation of the proteins isolated from the 40S subunits modified in the ternary (A,B) and binary (C) complexes; (D) typical stained gel, positions of the proteins are indicated according to (22). Positions of radioactive spots corresponding to the cross-linked proteins are marked with dotted lines.
Cross-linking of protein S26 to the mRNA in the ternary 80S ribosomal complexes was confirmed by immunoblotting; the data are presented in Figure 7 for analogues X–XII as examples. Antibodies raised against protein S26 produce an intense band corresponding to unmodified S26 (Figure 7B, all lanes) and a weaker band (lanes 1–3) whose position coincides with that of the intensive radioactive band in Figure 7A. The weaker band evidently corresponds to protein S26 cross-linked to the oligoribonucleotides. Protein S26 cross-linked to analogue XII has less electrophoretic mobility than that with analogues X and XI due to the larger molecular mass of the dodecamer XII as compared with the nonamers X and XI.
Figure 7. Identification of cross-linked S26 protein by immunoblotting after 1D-PAGE separation of 40S proteins with subsequent transfer onto a nitrocellulose membrane. Lanes 1, 2 and 3 correspond to the results with ternary complexes containing 80S ribosomes, tRNAPhe and analogue X, XI or XII, respectively. Lane K corresponds to a control experiment with proteins isolated from unmodified 40S subunits. (A) is an autoradiogram, (B) presents the identification of S26 with specific antibodies.
The results on cross-linking of mRNA analogues to ribosomal proteins are summarized in Table 2.
Table 2. Summary of the cross-linking data from positions –9 to +6 of mRNA on human 80S ribosomes obtained in this study
Identification of cross-linked nucleotides of 18S rRNA
RNA isolated from the irradiated ribosomal complexes was analysed by PAGE (data not shown). Cross-linking to the 18S rRNA was completely tRNA-dependent in all cases (no cross-linking occurs without tRNA). In ternary complexes, mRNA analogues cross-linked also to tRNA; this cross-linking was minor for the analogues II–VI and major for analogue I that modified tRNA more than 18S rRNA.
To determine the sequences of 18S rRNA containing cross-linked nucleotides, 18S rRNA isolated from irradiated complexes was digested with RNase H in the presence of deoxy-20mers complementary to various sequences of the 18S rRNA (from its 5'- to 3'-part) in parallel experiments. rRNA fragments resulting from hydrolysis were separated by denaturing PAGE, as in our previous studies (11,13,14). The results for all the mRNA analogues are summarized in Table 2.
The cross-linked nucleotides of 18S rRNA were identified by means of reverse transcription using primers chosen on the basis of the results of RNase H digestion (Figure 8). Primer extension leads to a stop or pause at the cross-linked rRNA nucleotide. The cross-linking site is generally assumed to be nucleotide 5' of the primer extension stop site. Therefore, the cross-linked 18S rRNA nucleotides were G626, G1702 and A1823–A1825. In particular, mRNA I cross-linked to nucleotide G1702 (Figure 8D), mRNAs II–VI to nucleotides A1823–A1825 (Figure 8A–C), and mRNAs V and VI also to G626 (Figure 8E). The stops are observed only for the respective ternary complexes of the ribosomes with the mRNA analogues and the cognate tRNA (lanes ‘+’). 18S rRNA samples modified with mRNA II and with mRNA I in the 80S ribosomal complex obtained with tRNAPhe were used here as controls since the rRNA nucleotides cross-linked with these mRNA analogues have been determined earlier (9,13). The results on the cross-linking of mRNA analogues to the 18S rRNA are summarized in Table 2.
Figure 8. Identification of sites of cross-linking of 18S rRNA to mRNA analogues by reverse transcription. Extension of 5'-32P-labelled primers complementary to 18S rRNA positions 1830–1849 (A–D) and 655–674 (E) on the 18S rRNA isolated from irradiated complexes. Lanes ‘–’ and ‘+’ correspond to the binary and ternary complexes, respectively. Lanes numbered ‘K’ are control 18S rRNA isolated from 80S ribosomes irradiated without mRNA analogues and treated in the same manner as the experimental samples. Lane marked ‘mRNA I*’ corresponds to 18S rRNA cross-linked with mRNA I in the 80S ribosomal complex obtained with tRNAPhe (guanosine with cross-linker at position +4). Lanes U, G, C, A, sequencing of 18S rRNA. Positions of the reverse transcription stops caused by the cross-linking are indicated.
DISCUSSION
mRNA analogues and model complexes
The design of the mRNA analogues was based on two main principles. First, the cross-linker was introduced at nucleotide positions that are not involved directly in Watson–Crick base pairing (C5 atoms of the uridines or N7 atoms of the guanos-ines). It had been previously shown that such modifications barely interfere with the coding properties of the triplets that contain the modified nucleotide (13). Second, binding of short mRNA analogues to 80S ribosomes is substantial only in the presence of a tRNA cognate to the selected mRNA codon that allows highly specific placement of the codon at the ribosomal P site. It should be noted that tRNAVal stimulated the binding of mRNA I much more weakly than the effect of tRNAPhe on the binding of mRNAs II–VI. This difference was caused by a higher affinity of tRNAPhe for its cognate codons UUU or UUC to the P site of the human 80S ribosome as compared with that of tRNAVal with its cognate codons (12). In general, the extent of cross-linking of mRNA analogue correlated with the level of its binding to the ribosomes.
Protein environment of mRNA on the ribosome
Positioning of mRNA with respect to 40S ribosomal proteins at the decoding site
In our earlier study (25), we reported that protein S15 cross-links to the derivative UUUGUU with a cross-linker on the guanosine residue when the latter is at position +1 or +4. Here, we have found this protein to cross-link with mRNA analogues bearing the cross-linker at the uridine residues if these are in positions from +4 to +6. For all these, the yield of the cross-link decreases significantly when the modified nucleotide moves from position +4 to position +6 (Figure 3). Therefore, we suggest that the first nucleotide of the A site codon is closest to the protein. Since nucleotides of the A site bound codon neighbour S15 and the 18S rRNA nucleotides A1823–A1825 and G626, it is reasonable to suggest that these rRNA nucleotides are in the vicinity of S15 at the decoding site of human 80S ribosomes. Cross-linking to S3 (together with other proteins) was detected previously when the modified uridine residues of the mRNA analogues, derivatives of UUUGUU, were at the P or E site (positions from –3 to +3) (10), and also for alkylating mRNA analogues when the modified nucleotide was at positions –6, –3, +6/+7 or +12/+13 (12). Protein S3 also cross-links to mRNA analogues without tRNA within labile binary complexes with 80S ribosomes, poorly detectable by the nitrocellulose filtration technique. This cross-linking actually occurs at the mRNA-binding site since it is inhibited by the addition of poly(U) (data not shown). Evidently, a site of tRNA-independent binding of mRNA analogues on the 80S ribosomes lies close to protein S3. Based on the data discussed one can suggest that in contrast to S15, protein S3 is close to the mRNA in various parts of the mRNA-binding channel of the 80S ribosomes, including its regions distant from the decoding site.
Arrangement of the upstream mRNA region
Modified mRNA nucleotides cross-link only to 40S ribosomal proteins from positions –4 to –9. The cross-linking patterns are almost insensitive to the nature of the nucleotide and the position of the nucleotide regarding the P site bound codon within the interval –4 to –9. This indicates that the upstream mRNA region is surrounded mainly by proteins on the 80S ribosome. The sets of proteins cross-linked to the modified uridines and guanosines from positions –4 to –9 are very similar, and the main target for cross-linking in all cases is S26. The same protein was earlier found to cross-link from mRNA positions +1 and –3 (10); thus, S26 is close to at least 10 mRNA nucleotides at positions from –9 to +1. Earlier, using alkylating mRNA analogues it was reported that the reactive group in the 5'-terminal nucleotide at positions from –6 to +12/13 cross-linked to proteins S3 and S3a (20). The difference is evidently due to the different natures of the alkylating and photoreactive cross-linkers in the mRNA analogues that may result in the formation of different modification products .
In addition, minor cross-linking to protein S14 from position –6 (see the data for analogues VIII and XI in Figures 2 and 6) points to the possible proximity between this protein and the mRNA at nucleotide position –6. It is worthy of notice that it was proposed on the basis of the cryoelectron microscopy data of mammalian ribosomes that S14 is located close to the mRNA exit tunnel (26) where the 5'-terminal sequence of the ribosome-bound portion of the mRNA is positioned. In binary complexes with 80S ribosomes, mRNA analogues VII–XII cross-link to protein S3 as do analogues II–VI (see above). Clearly, without tRNA short mRNA analogues bind to 80S ribosomes at a site close to protein S3.
Positioning of mRNA with respect to 18S rRNA at the decoding site
The +1/ G1702 cross-link was found for the first time in this study. This cross-link is relatively weak since tRNAVal bound at the P site (Figure 1, complex 1) shields 18S rRNA from cross-linking. This assumption is confirmed by the data on cross-linking of mRNA I to 18S rRNA in 80S ribosomal complexes where the modified GUU codon was bound to the A site and the cross-linker was in position +4 (13). In this case, the binding of a tRNA molecule in the A site resulted in significant decrease of cross-linking of the mRNA analogue to nucleotides A1823 and A1824 of the 18S rRNA.
Earlier it was shown that mRNA analogues with a perfluoro-phenylazide group at either the 5'-phosphate or the C5 atom of the 5'-terminal uridine at position +1 cross-linked to nucleotide G1207 of the 18S rRNA (13,14). In the present study, the same cross-linker at the N7 atom of the guanosine in the middle part of the mRNA analogue modified G1702, and no cross-linking was found in other regions of the 18S rRNA. These differences seem to be caused by different spatial orientations of the cross-linkers. Cross-linking of different modified mRNA nucleotides from position +1 to nucleotides G1207 (in the middle of helix 28) and G1702 (at the beginning of helix 44) is not surprising since these nucleotides are located close to each other in the secondary structure of the 18S rRNA (see www.rna.utexas.edu). It was previously reported that nucleotide G1702 cross-linked to the 3'-terminal alkylating group of mRNA analogues when the corresponding 3'-terminal nucleotide was at positions +3/+4 and +6/+7 (12). Cross-linking from positions +1, +3/+4 and +6/+7 to the same nucleotide G1702 of 18S rRNA may reflect the existence of a turn between codons bound at the A and P sites of the 80S ribosome.
In our previous reports (9,13) using mRNAs I and II, we showed that 18S rRNA nucleotides A1823 and A1824 are close to the first nucleotide of the codon bound to the A site of human ribosomes. The results of the present study for the first time show that all nucleotides of the A site bound codon are in the vicinity of nucleotides A1823–A1825 of the 18S rRNA. Moreover, we found that the second and third nucleotides of this codon are also close to G626. The latter was cross-linked only in the case of the guanosine-modified mRNA analogues V and VI. The lack of cross-linking of G626 with the modified uridine residues at the same positions (mRNAs III and IV) may be explained by different geometries of the spacers that link the photoactivatable moieties to the uridine and the guanosine residues (Figure 1). A cross-linker at the uridine may be unable to reach G626.
Extent of conservation of the mRNA-binding track on the ribosome
Location of template on the human ribosome
The results of this study together with previous cross-linking data on human ribosomes (9–11,13,20) provide a view on the positioning of the mRNA on the human ribosome. According to the scheme presented in Figure 9, nucleotide G961 of the 18S rRNA is in the vicinity of mRNA positions –3 and –2; nucleotide G1207 is close to mRNA positions from –3 to +3, nucleotide G1702 is close to the P site bound codon (position +1 to +3), nucleotides A1823–A1825 are in the proximity of the A site bound codon (positions +4 to +6), and nucleotide G626 is close to two nucleotides of the A site bound codon (positions +5 and +6). Protein S26 surrounds the mRNA positions from –9 to +1, whereas S15 is in the vicinity of P and A site bound codons (positions +1 to +6). Protein S3a most probably neighbours the long mRNA region including a portion the upstream region, the region of the codon–anticodon interactions and a portion of the downstream region.
Figure 9. Scheme of the mutual location of a template and the structural elements of the 40S subunit in the region of the mRNA-binding site of human ribosomes. Large circles, ribosomal proteins; small circles, nucleotides of 18S rRNA.
Variability of protein environment of mRNA on the ribosome
Cryo-electron microscopy data on eukaryotic ribosomes show that the structures of the active sites of pro- and eukaryotic ribosomes are in general similar (26,27). Nevertheless, a comparison of the tRNA-dependent cross-links of human 80S ribosomes with the X-ray crystallographic data obtained with prokaryotic ribosomes (1–4) does not reveal visible similarities in the proteins neighbouring the A site bound codon between prokaryotic and mammalian ribosomes. In the prokaryotic ribosome, only protein S12 is located close to the decoding site. Eukaryotic protein S23, the homologue of the prokaryotic S12 protein (28), did not cross-link to any mRNA analogue in the present study. According to our cross-linking data, all the nucleotides of the A site codon lie close to protein S15 on the 80S ribosome. Its prokaryotic homologue S19 (28), located mainly on the head of the 30S subunit away from the mRNA track has a tail that extends towards the decoding site (1,4). However, the minimum distance between the tail and the A site bound codon calculated from models (1,4) is between 39 ? (position +4) and 45 ? (position +6). If the spatial structure of mammalian S15 in the 40S subunit is similar to that of S19 in the 30S subunit (i.e. minimum distance between the tail of S15 and the A site bound codon is 40 ?), a cross-linker with a length of no more than 11 ? attached to nucleotides of the A site bound codon could not reach the tail of S15. The position of the main globular part of S15 on the head of the 40S subunit (27) is similar to that of S19 on the head of the 30S subunit (29). Based on the results of this study, we suggest that the tail of mammalian S15 in the 40S subunit comes substantially closer to the decoding site than the tail of prokaryotic S19 in the 30S subunit.
Other more distinct differences concern the environment of the mRNA region upstream of the E site bound codon (positions –9 to –4). First, mammalian proteins S26 (this study) and S3a (12,20) that have no homologues among prokaryotic ribosomal proteins (28) predominantly surround this mRNA region . Second, the 18S rRNA was not found in the molecular environment of the upstream part of the mRNA. In contrast, according to X-ray crystallographic data on prokaryotic 70S ribosomes (4), the immediate molecular environment of mRNA positions –9 to –4 contains mainly 16S rRNA, and protein S11 interacts with the backbone of the mRNA around positions –6 to –4. Data on the spatial structures of eukaryotic proteins S3a and S26 and the location of these proteins on the 40S subunit are not available; therefore it is difficult to conclude which parts of the proteins lie close to the mRNA upstream of the E site bound codon. However, it seems more probable that the upstream part of the mRNA is close to the main part of S26 since a long stretch of mRNA from positions –9 to +1 was found to neighbour this protein. One can suggest that S26 shields 18S rRNA and protein S14 from contacts with the upstream part of the mRNA.
Conservation of positioning of mRNA at the decoding site of the small subunit rRNA
Nucleotides A1824, A1825, G1702 and G626 of the 18S rRNA are conserved in the secondary structure of the small rRNA subunit and correspond to A1492, A1493, G1401 and G530 of the prokaryotic 16S rRNA (see www.rna.utexas.edu and Figure 10). It is known that nucleotides A1492 and A1493 of 16S rRNA are involved in the decoding of prokaryotic ribosomes and play a key role in the discrimination of correct codon–anticodon duplex at the A site (31). According to X-ray crystallographic data (1,3,4), nucleotides A1492, A1493, G1401 and G530 of 16S rRNA participate directly in the accommodation of mRNA codons in the region of codon–anticodon interactions on prokaryotic ribosomes. Thus, the phosphate of G1401 is located between the third nucleotide of the P site codon and the first nucleotide of the A site codon and produces a kink in the mRNA between the A and P site codons. Nucleotides A1492, A1493 and G530 are involved in the formation of the decoding site. A1493 facilitates fixation of the first pair of the codon–anticodon duplex at the A site by hydrogen bonds between the first nucleotide of the codon and the last nucleotide of the anticodon. A1492 interacts with the second nucleotide of the A site codon, and G530 interacts with the second nucleotide of the anticodon; G530 also takes part in the fixation of third pair in the codon–anticodon duplex (3,4). The results on human ribosomes clearly are in remarkable agreement with X-ray crystallographic data on prokaryotic ribosomes. Consequently, the data obtained reveal the highly conserved nature of the decoding site of the small subunit rRNA for all classes of ribosomes.
Figure 10. Fragment of 18S rRNA secondary structure (from www.rna.utexas.edu) containing conserved nucleotides capable of cross-linking to various positions of mRNA analogues (indicated in boxes). The corresponding nucleotides of E.coli 16S rRNA are numbered in brackets.
Concluding remarks
Our study experimentally confirms the widely accepted idea on the conserved rRNA ‘core’ of the ribosome that appears to be structurally similar in all kingdoms. At the same time, our findings indicate noticeable dissimilarities in the molecular environment of the mRNA in pro- and eukaryotic ribosomes. The first concerns proteins neighbouring the decoding site and seems to be caused by different positioning of the tails of mammalian S15 and its prokaryotic homologue S19 with respect to this site. Other more important differences are related to the molecular environment of the mRNA region upstream of the E site bound codon which on 80S ribosomes is surrounded largely by proteins, in contrast to 70S ribosomes in which the environment of this part of the mRNA consists of both rRNA and proteins. The proteins surrounding the upstream part of the mRNA on the 80S ribosome have no prokaryotic homologues. The latter differences may be related to the more complicated process of translation regulation in eukaryotes as compared with that in prokaryotes. Probably, the binding site of the upstream part of the mRNA on the 80S ribosome is formed by eukaryotic-specific proteins that may be targets for binding of various regulatory factors (such as chaperones, modifying enzymes, etc.) capable of affecting the accuracy and rate of translation. Future structural and biochemical experiments will be required to address these aspects of the complicated mechanisms used by the eukaryotic ribosomal machinery.
ACKNOWLEDGEMENTS
We gratefully thank Anne-Lise Haenni and Richard Brimacombe for critical reading of this manuscript. This work was supported by the Russian Foundation for Basic Research (grant no. 02-04-48194 to G.K.).
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