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1.
Models of the atomic structure of the eukaryotic translation termination complex containing mRNA, P-site tRNAPhe, human class 1 release factor eRF1, and 80S ribosome, were constructed by computational modeling. The modeling was based on the assumed structural-functional similarity between the tRNA and eFR1 molecules in the ribosomal A site. The known atomic structure of the 70S ribosome complexed with mRNA as well as the P-and A-site tRNAsPhe was used as a structural template for the modeling. The eRF1 molecule bound in the A site undergoes substantial conformational changes so that the mutual configuration of the N and M domains matches the overall tRNA shape. Two models of eRF1 binding to mRNA at the A site in the presence of P-site tRNAPhe were generated. A characteristic of these models is complementary interactions between the mRNA stop codon and the grooves at different sides of the surface of the eRF1 fragment, containing helix α2, NIKS loop, and helix α3 of the N domain. In model 1, the nucleotides of the mRNA stop codon at the A site are approximately equidistant (~15 Å) from the N (motifs NIKS and YxCxxxF) and C domains. In model 2, the stop codon is close to the N-domain motifs NIKS and YxCxxxF. Both models fit genetic and biochemical experimental data. The choice of a particular model requires additional studies. 相似文献
2.
A properties of atomic models of structure of eukaryotic triple complex eRF1 . mRNA . tRNAPhe containing human class-1 polypeptide release factor eRF1 at the A-site of human 80S ribosome, mRNA and P-site tRNAPhe, obtained before, are considered. The stricture of the complex is described using high resolution NMR structure of eRF1 M-domain. The structural properties of distribution of chemical cross-links are investigated, which allows us to choose correct model of positioning of the eRF1 molecule in ribosome A-site relative to stop codon of mRNA. A distributions of crosslinks between photoactivatable perfluoroaryl azide group of modified nucleotides of mRNA analogues and eRF1 molecule are modeled via molecular dynamics method. Twelve different mRNA analogues with modified nucleotides of stop signal in positions +4 to +9 with respect to the first nucleotide of the P-site codon are modeled. It was shown that only one of the two models of complex eRFI . mRNA . tRNA gives cross-link distribution in a good agreement with experimental data. A new features of the final structure of triple complex eRF1 . mRNA . tRNA is spatial proximity of stop-codon nucleotides to the C-domain of the eRF1, which explains previously obtained cross-link experimental data. 相似文献
3.
K. N. Bulygin E. A. Popugaeva M. N. Repkova M. I. Meschaninova A. G. Ven’yaminova D. M. Graifer L. Yu. Frolova G. G. Karpova 《Molecular Biology》2007,41(5):781-789
The arrangement of the stop codon and its 3′-flanking codon relative to the components of translation termination complexes of human 80S ribosomes was studied using mRNA analogs containing the stop signal UPuPuPu (Pu is A or G) and the photoreactive perfluoroarylazido group, which was linked to a stop-signal or 3′-flanking nucleotide (positions from +4 to +9 relative to the first nucleotide of the P-site codon). Upon mild UV irradiation, the analogs crosslinked to components of the model complexes, mimicking the state of the 80S ribosome at translation termination. Termination factors eRF1 and eRF3 did not change the relative arrangement of the stop signal and 18S rRNA. Crosslinking to eRF1 was observed for modified nucleotides in positions +5 to +9 (that for stop-codon nucleotide +4 was detected earlier). The eRF1 fragments crosslinked to the mRNA analogs were identified. Fragment 52–195, including the N domain and part of the M domain, crosslinked to the analogs carrying the reactive group at A or G in positions +5 to +9 or at the terminal phosphate of nucleotide +7. The site crosslinking to mRNA analogs containing modified G in positions +5 to +7 was assigned to eRF1 fragment 82–166 (beyond the NIKS motif). All but one analog (that with modified G in position +4) crosslinked to the C domain of eRF1 (fragment 330–422). The efficiency of crosslinking to the C domain was higher than to the N domain in most cases. It was assumed that the C domain of eRF1 bound in the A site is close to nucleotides +5 to +9, especially +7 and +8, and that eRF1 undergoes substantial conformational changes when binding to the ribosome. 相似文献
4.
How are tRNAs and mRNA arranged in the ribosome? An attempt to correlate the stereochemistry of the tRNA-mRNA interaction with constraints imposed by the ribosomal topography. 总被引:3,自引:0,他引:3
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V Lim C Venclovas A Spirin R Brimacombe P Mitchell F Müller 《Nucleic acids research》1992,20(11):2627-2637
Two tRNA molecules at the ribosomal A- and P-sites, with a relatively small angle between the planes of the L-shaped molecules, can be arranged in two mutually exclusive orientations. In one (the 'R'-configuration), the T-loop of the A-site tRNA faces the D-loop of the P-site tRNA, whereas in the other (the 'S'-configuration) the D-loop of the A-site tRNA faces the T-loop of the P-site tRNA. A number of stereochemical arguments, based on the crystal structure of 'free' tRNA, favour the R-configuration. In the ribosome, the CCA-ends of the tRNA molecules are 'fixed' at the base of the central protuberance (the peptidyl transferase centre) of the 50S subunit, and the anticodon loops lie in the neck region (the decoding site) of the 30S subunit. The translocation step is essentially a rotational movement of the tRNA from the A- to the P-site, and there is convincing evidence that the A-site must be located nearest to the L7/L12 protuberance of the 50S subunit. The mRNA in the two codon-anticodon duplexes lies on the 'inside' of the 'elbows' of the tRNA molecules (in both the S-type and R-type configurations), and runs up between the two molecules from the A- to the P-site in the 3' to 5'-direction. These considerations have the consequence that in the S-configuration the mRNA in the codon-anticodon duplexes is directed towards the 50S subunit, whereas in the R-configuration it is directed towards the 30S subunit. The results of site-directed cross-linking experiments, in particular cross-links to mRNA at positions within or very close to the codons interacting with A- or P-site tRNA, favour the latter situation. This conclusion is in direct contradiction to other current models for the arrangement of mRNA and tRNA on the ribosome. 相似文献
5.
The integral structural parameters and the shape of the molecule of human translation termination factor eRF1 were determined from the small-angle X-ray scattering in solution. The molecular shapes were found by bead modeling with nonlinear minimization of the root-mean-square deviation of the calculated from the experimental scattering curve. Comparisons of the small-angle scattering curves computed for atomic-resolution structures of eRF1 with the experimental data on scattering from solution testified that the crystal and the solution conformations are close. In the ribosome, the distance between the eRF1 motifs GGQ and NIKS must be shorter than in crystal or solution (75 versus 100–107 Å). Therefore, like its bacterial counterpart RF2, the eukaryotic eRF1 must change its conformation as it binds to the ribosome. The conformational mobility of eukaryotic and prokaryotic class-1 release factors is another feature making them functionally akin to tRNA. 相似文献
6.
The invariant uridine of stop codons contacts the conserved NIKSR loop of human eRF1 in the ribosome
To unravel the region of human eukaryotic release factor 1 (eRF1) that is close to stop codons within the ribosome, we used mRNAs containing a single photoactivatable 4-thiouridine (s(4)U) residue in the first position of stop or control sense codons. Accurate phasing of these mRNAs onto the ribosome was achieved by the addition of tRNA(Asp). Under these conditions, eRF1 was shown to crosslink exclusively to mRNAs containing a stop or s(4)UGG codon. A procedure that yielded (32)P-labeled eRF1 deprived of the mRNA chain was developed; analysis of the labeled peptides generated after specific cleavage of both wild-type and mutant eRF1s maps the crosslink in the tripeptide KSR (positions 63-65 of human eRF1) and points to K63 located in the conserved NIKS loop as the main crosslinking site. These data directly show the interaction of the N-terminal (N) domain of eRF1 with stop codons within the 40S ribosomal subunit and provide strong support for the positioning of the eRF1 middle (M) domain on the 60S subunit. Thus, the N and M domains mimic the tRNA anticodon and acceptor arms, respectively. 相似文献
7.
Affinity labelling of E. coli ribosomes near the donor tRNA-binding (P) site was studied with the use of photoreactive derivatives of tRNAPhe bearing arylazidogroups on N7 atoms of guanine residues (azido-tRNA). UV-irradiation of complexes 70S ribosome.poly(U).azido- tRNA(P-site) and 70S ribosome.poly(U).azido-tRNA(P-site).Phe- tRNAPhe(A-site) resulted in covalent attachment of azido-tRNA to ribosomes, both subunits being labelled. In both cases modification extent of 30S subunit was two-fold than that of the 50S one. It was shown that when the A-site was free the azido-tRNA located in P-site labelled proteins S9, S11, S12, S13, S21 and L14, L27, L31. Azido-tRNA located in P-site when the A-site was occupied with Phe-tRNAPhe labelled proteins S11, S12, S13, S14, S19, L32/L33 and possibly L23, L25. From the comparison of the sets of proteins labelled when A-site was free or occupied a conclusion was drawn that aminoacyl-tRNA located in ribosomal A-site affects the arrangement of deacylated tRNA in P-site. Data obtained allow to propose that proteins S5, S19, S20 and L24, L33 interact with guanine residues important for the tRNA tertiary structure formation. 相似文献
8.
The integral structural parameters and the shape of the molecule of human translation termination factor eRF1 were determined from the small-angle X-ray scattering in solution. The molecular shapes were found by bead modeling with nonlinear minimization of the root-mean-square deviation of the calculated from the experimental scattering curve. Comparisons of the small-angle scattering curves computed for atomic-resolution structures of eRF1 with the experimental data on scattering from solution testified that the crystal and the solution conformations are close. In the ribosome, the distance between the eRF1 motifs GGQ and NIKS must be shorter than in crystal or solution (75 versus 107-112 A). Therefore, like its bacterial counterpart RF2, the eukaryotic eRF1 must change its conformation as it binds to the ribosome. The conformational mobility of eukaryotic and prokaryotic class-1 release factors is another feature making them functionally akin to tRNA. 相似文献
9.
EF-G-catalyzed translocation of anticodon stem-loop analogs of transfer RNA in the ribosome. 总被引:3,自引:1,他引:2
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Translocation, catalyzed by elongation factor EF-G, is the precise movement of the tRNA-mRNA complex within the ribosome following peptide bond formation. Here we examine the structural requirement for A- and P-site tRNAs in EF-G-catalyzed translocation by substituting anticodon stem-loop (ASL) analogs for the respective tRNAs. Translocation of mRNA and tRNA was monitored independently; mRNA movement was assayed by toeprinting, while tRNA and ASL movement was monitored by hydroxyl radical probing by Fe(II) tethered to the ASLs and by chemical footprinting. Translocation depends on occupancy of both A and P sites by tRNA bound in a mRNA-dependent fashion. The requirement for an A-site tRNA can be satisfied by a 15 nucleotide ASL analog comprising only a 4 base pair (bp) stem and a 7 nucleotide anticodon loop. Translocation of the ASL is both EF-G- and GTP-dependent, and is inhibited by the translocational inhibitor thiostrepton. These findings show that the D, T and acceptor stem regions of A-site tRNA are not essential for EF-G-dependent translocation. In contrast, no translocation occurs if the P-site tRNA is substituted with an ASL, indicating that other elements of P-site tRNA structure are required for translocation. We also tested the effect of increasing the A-site ASL stem length from 4 to 33 bp on translocation from A to P site. Translocation efficiency decreases as the ASL stem extends beyond 22 bp, corresponding approximately to the maximum dimension of tRNA along the anticodon-D arm axis. This result suggests that a structural feature of the ribosome between the A and P sites, interferes with movement of tRNA analogs that exceed the normal dimensions of the coaxial tRNA anticodon-D arm. 相似文献
10.
Locking and unlocking of ribosomal motions 总被引:20,自引:0,他引:20
During the ribosomal translocation, the binding of elongation factor G (EF-G) to the pretranslocational ribosome leads to a ratchet-like rotation of the 30S subunit relative to the 50S subunit in the direction of the mRNA movement. By means of cryo-electron microscopy we observe that this rotation is accompanied by a 20 A movement of the L1 stalk of the 50S subunit, implying that this region is involved in the translocation of deacylated tRNAs from the P to the E site. These ribosomal motions can occur only when the P-site tRNA is deacylated. Prior to peptidyl-transfer to the A-site tRNA or peptide removal, the presence of the charged P-site tRNA locks the ribosome and prohibits both of these motions. 相似文献
11.
Yu. S. Khairulina M. V. Molotkov K. N. Bulygin D. M. Graifer A. G. Ven’yaminova L. Yu. Frolova J. Stahl G. G. Karpova 《Russian Journal of Bioorganic Chemistry》2008,34(6):691-697
Protein S3 fragments were determined that crosslink to modified mRNA analogues in positions +5 to +12 relative to the first nucleotide in the P-site bound codon in model complexes mimicking states of ribosomes at the elongation and translation termination steps. The mRNA analogues contained a Phe codon UUU/UUC at the 5′-termini that could predetermine the position of the tRNAPhe on the ribosome by the P-site binding and perfluorophenylazidobenzoyl group at a nucleotide in various positions 3′ of the UUU/UUC codon. The crosslinked S3 protein was isolated from 80S ribosomal complexes irradiated with mild UV light and subjected to cyanogen bromide—induced cleavage at methionine residues with subsequent identification of the crosslinked oligopeptides. An analysis of the positions of modified oligopeptides resulting from the cleavage showed that, in dependence on the positions of modified nucleotides in the mRNA analogue, the crosslinking sites were found in the N-terminal half of the protein (fragment 2–217) and/or in the C-terminal fragment 190–236; the latter reflects a new peculiarity in the structure of the mRNA binding center in the ribosome, unknown to date. The results of crosslinking did not depend on the type of A-site codon or on the presence of translation termination factor eRF1. 相似文献
12.
To add a tag-peptide for degradation to the nascent polypeptide in a stalled ribosome, an unusual translation called trans-translation is facilitated by transfer-messenger RNA (tmRNA) having an upper half of the tRNA structure and the sequence encoding the tag-peptide except the first alanine. During this event, tmRNA enters the vacant A-site of the stalled ribosome without a codon–anticodon interaction, but with a protein factor SmpB. Here, we studied the sites and modes of binding of SmpB to the ribosome by directed hydroxyl radical probing from Fe(II) tethered to SmpB variants. It revealed two SmpB-binding sites, A-site and P-site, on the ribosome. Each SmpB can be superimposed on the lower half of tRNA behaving in translation. The sites of cleavages from Fe(II) tethered to the C-terminal residues of A-site SmpB are aligned along the mRNA path towards the downstream tunnel, while those of P-site SmpB are found almost exclusively around the region of the codon–anticodon interaction in the P-site. We propose a new model of trans-translation in that the C-terminal tail of SmpB initially recognizes the decoding region and the mRNA path free of mRNA by mimicking mRNA. 相似文献
13.
The effect of tRNA binding on the structure of 5 S RNA in Escherichia coli. A chemical modification study 总被引:5,自引:0,他引:5
The structure of 5 S RNA within the 70 S ribosome from Escherichia coli was studied using the chemical reagent kethoxal (alpha-keto-beta-ethoxybutyraldehyde) to modify accessible guanosines. The modification pattern of 5 S RNA from free 70 S ribosomes was compared with that of poly(U) programmed ribosomes where tRNA had been bound to both the A- and P-sites. Binding to the ribosomal A-site was achieved enzymatically using the elongation factor Tu and GTP in the presence of deacylated tRNA which blocks the ribosomal P-site. Modified guanosines were identified after partial RNase T1 hydrolysis and separation of the hydrolysis products on sequencing gels. Binding of tRNA to the ribosome leads to a strong protection of 5 S RNA guanosine G-41 and to some degree G-44 from kethoxal modification. The limited RNase T1 hydrolysis pattern provides evidence for the existence of a 5 S RNA conformation different from the known 5 S RNA A- and B-forms which are characterized by their gel electrophoretic mobility. The importance of 5 S RNA for the binding of tRNA to the ribosome is discussed. 相似文献
14.
A consensus has been reached that the conformation of the anticodon-codon interactions of two adjacent tRNA molecules on the ribosome is a Sundaralingam-type (S-type). Even if it is kept to the S-type, there are still various possibilities. Various experimental data have been supporting an idea that the conformation of A-site tRNA is different from that of P-site tRNA. Those data as well as the recent result of Brimacombe and co-workers that U20:1 of lupin tRNAmMetbound to the A-site was cross-linked to a region, 875-905, of 23S rRNA in combination with the other recent findings of Nierhaus and co-workers about the spin-contrast method of neutron diffraction of the ribosome and the better accessible nucleotide patterns of phosphorothioated tRNAs on the ribosome have led to a new tRNA docking pair model, in which the highly conserved G18 and G19 of D-loop in A-site tRNA and C56 and C61 of TpsiC-loop in P-site tRNA base pair along with the conventional base pairs of adjacent codon-anticodon interactions. This A-P tRNA pair model can be translocated to the P-E tRNA pair model without changing the conformation except the ACCA termini, keeping the position of the growing nascent polypeptide chain. 相似文献
15.
G T Babkina S N Vladimirov S A Dryga M A Zenkova G G Karpova 《Molekuliarnaia biologiia》1988,22(2):357-361
Affinity labeling of E. coli ribosomes with the 2',3'-O-[4-(N-2-chloroethyl)-N-methyl-amino]benzylidene derivative of AUGU6 (AUGU6-[14C]CHRCl) was studied within the pretranslocational complex ribosome.AUGU6[14C]CHRCl.tRNA(fMet)(P-site).fMetPhe-tR NA(Phe)(A-site) and posttranslocational complex ribosome.AUGU6[14C]CHRCl.fMetPhe-tRNA(Phe)(P-site). Both 30S and 50S subunits were labeled within these complexes, but the extent of 30S subunit modification was 6-8-fold higher than those for 50S subunit. Ribosomal proteins of both subunits were found to be labeled preferentially. Proteins S1, S5, S11, L1 were identified to be crosslinked with AUGU6[14C]CHRCl within the pretranslocational complex and S7--within the posttranslocational complex from the data of two-dimensional electrophoresis in the polyacrylamide gel. 相似文献
16.
A study was made of the properties of the two structural models that had previously been constructed for the eukaryotic triple complex eRF1 · mRNA · tRNAPhe with eRF1 accommodated in the A site and tRNAPhe, in the P site of the ribosome. The structure of the complex was described using a high-resolution NMR structure of the human eRF1 M domain. The distribution of chemical crosslinks between mRNA and eRF1 was studied for the two models, which made it possible to decide about the positioning of eRF1 in the A site relative to the mRNA stop codon. Molecular dynamics was used to simulate the distribution of close contacts (<7 Å) between the photoactivatable azido group of modified mRNA analogs and eRF1 residues in the complex. Analysis of the structures of 12 analogs containing a modified nucleotide with the photoactivatable group in a position from +4 to +9 showed that only one model of eRF1 binding with mRNA in the A site well agreed with experimental data on chemical crosslinking. A new feature of the model selected is that the C domain of eRF1 is close to the mRNA stop-codon nucleotides, which explained the experimental findings. 相似文献
17.
Sergiev PV Lesnyak DV Kiparisov SV Burakovsky DE Leonov AA Bogdanov AA Brimacombe R Dontsova OA 《Nucleic acids research》2005,33(18):6048-6056
Ribosomes synthesize proteins according to the information encoded in mRNA. During this process, both the incoming amino acid and the nascent peptide are bound to tRNA molecules. Three binding sites for tRNA in the ribosome are known: the A-site for aminoacyl-tRNA, the P-site for peptidyl-tRNA and the E-site for the deacylated tRNA leaving the ribosome. Here, we present a study of Escherichia coli ribosomes with the E-site binding destabilized by mutation C2394G of the 23S rRNA. Expression of the mutant 23S rRNA in vivo caused increased frameshifting and stop codon readthrough. The progression of these ribosomes through the ribosomal elongation cycle in vitro reveals ejection of deacylated tRNA during the translocation step or shortly after. E-site compromised ribosomes can undergo translocation, although in some cases it is less efficient and results in a frameshift. The mutation affects formation of the P/E hybrid site and leads to a loss of stimulation of the multiple turnover GTPase activity of EF-G by deacylated tRNA bound to the ribosome. 相似文献
18.
Konstantin N. Bulygin Yulia S. Khairulina Petr M. Kolosov Aliya G. Ven'yaminova Dmitri M. Graifer Yuri N. Vorobjev Ludmila Yu. Frolova Lev L. Kisselev Galina G. Karpova 《RNA (New York, N.Y.)》2010,16(10):1902-1914
To study positioning of the polypeptide release factor eRF1 toward a stop signal in the ribosomal decoding site, we applied photoactivatable mRNA analogs, derivatives of oligoribonucleotides. The human eRF1 peptides cross-linked to these short mRNAs were identified. Cross-linkers on the guanines at the second, third, and fourth stop signal positions modified fragment 31–33, and to lesser extent amino acids within region 121–131 (the “YxCxxxF loop”) in the N domain. Hence, both regions are involved in the recognition of the purines. A cross-linker at the first uridine of the stop codon modifies Val66 near the NIKS loop (positions 61–64), and this region is important for recognition of the first uridine of stop codons. Since the N domain distinct regions of eRF1 are involved in a stop-codon decoding, the eRF1 decoding site is discontinuous and is not of “protein anticodon” type. By molecular modeling, the eRF1 molecule can be fitted to the A site proximal to the P-site-bound tRNA and to a stop codon in mRNA via a large conformational change to one of its three domains. In the simulated eRF1 conformation, the YxCxxxF motif and positions 31–33 are very close to a stop codon, which becomes also proximal to several parts of the C domain. Thus, in the A-site-bound state, the eRF1 conformation significantly differs from those in crystals and solution. The model suggested for eRF1 conformation in the ribosomal A site and cross-linking data are compatible. 相似文献
19.
Hirokawa G Kiel MC Muto A Selmer M Raj VS Liljas A Igarashi K Kaji H Kaji A 《The EMBO journal》2002,21(9):2272-2281
Ribosome recycling factor (RRF) together with elongation factor G (EF-G) disassembles the post- termination ribosomal complex. Inhibitors of translocation, thiostrepton, viomycin and aminoglycosides, inhibited the release of tRNA and mRNA from the post-termination complex. In contrast, fusidic acid and a GTP analog that fix EF-G to the ribosome, allowing one round of tRNA translocation, inhibited mRNA but not tRNA release from the complex. The release of tRNA is a prerequisite for mRNA release but partially takes place with EF-G alone. The data are consistent with the notion that RRF binds to the A-site and is translocated to the P-site, releasing deacylated tRNA from the P- and E-sites. The final step, the release of mRNA, is accompanied by the release of RRF and EF-G from the ribosome. With the model post-termination complex, 70S ribosomes were released from the post-termination complex by the RRF reaction and were then dissociated into subunits by IF3. 相似文献
20.
VanLoock MS Agrawal RK Gabashvili IS Qi L Frank J Harvey SC 《Journal of molecular biology》2000,304(4):507-515
The ribosome undergoes pronounced periodic conformational changes during protein synthesis. Of particular importance are those occurring around the decoding site, the region of the 16 S rRNA interacting with the mRNA-(tRNA)(2) complex. We have incorporated structural information from X-ray crystallography and nuclear magnetic resonance into cryo-electron microscopic maps of ribosomal complexes designed to capture structural changes at the translocation step of the polypeptide elongation cycle. The A-site region of the decoding site actively participates in the translocation of the tRNA from the A to the P-site upon GTP hydrolysis by elongation factor G, shifting approximately 8 A toward the P-site. This implies that elongation factor G actively pushes both the decoding site and the mRNA/tRNA complex during translocation. 相似文献