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ABSTRACT: 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.
Molecular Biology 09/2007; 41(5):781-789. · 0.66 Impact Factor
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ABSTRACT: The protein environment of mRNA 3′ of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set
of oligoribonucleotide derivatives bearing a UUU triplet at the 5′-end and a perfluoroarylazide group at one of the nucleotide
residues 3′ of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNAPhe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNAPhe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with
proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target
for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the
S3 and S2 proteins occurred both in ternary complexes and in the absence of tRNA. Within ternary complexes, crosslinking with
S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to
+12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed; it
was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that
the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3′ of the codon in the decoding
site.
Russian Journal of Bioorganic Chemistry 06/2007; 33(4):399-409. · 0.64 Impact Factor
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ABSTRACT: The protein environment of mRNA 3' of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5'-end and a perfluoroarylazide group at one of the nucleotide residues at the 3'-end of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNAPhe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNAPhe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in triple complexes and in the absence of tRNA. Within triple complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed, it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3' of the codon in the decoding site.
Bioorganicheskaia khimiia 33(4):431-41.
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ABSTRACT: Positioning of stop codon and the adjacent triplet downstream of it with respect to the components of human 80S termination complex was studied with the use of mRNA analogues that bore stop signal UPuPuPu (Pu is A or G) and photoactivatable perfluoroaryl azide group. This group was attached to one of nucleotides of the stop signal or 3' of it (in positions +4 to +9 with respect to the first nucleotide of the P site codon). It was shown that upon mild UV irradiation the mRNA analogues crosslinked to components of model complexes imitating state of 80S ribosome in the course of translation termination. It was found that termination factors eRF1 and eRF3 do not affect mutual arrangement of stop signal and the 18S rRNA. Factor eRF1 was shown to cross-link to modified nucleotides in positions +5 to +9 (ability of eRF1 to cross-link to stop codon nucleotide in position +4 was shown earlier). Fragments of eRF1 containing cross-linking sites of the mRNA analogues were determined. In fragment 52-195 (containing the N-domain and a part of the M-domain) we have found cross-linking sites of the analogues that bore modifying groups on A or G in positions +5 to +9 or at the terminal phosphate of nucleotide in position +7. For mRNA analogues bearing modifying groups on G site of cross-linking from positions +5 to +7 was found in the eRF1 fragment
Molekuliarnaia biologiia 41(5):858-67.
