The polypeptide chain release factor eRF1 specifically contacts the s(4)UGA stop codon located in the A site of eukaryotic ribosomes.
It has been shown previously [Brown, C.M. & Tate, W.P. (1994) J. Biol. Chem. 269, 33164-33170.] that the polypeptide chain release factor RF2 involved in translation termination in prokaryotes was able to photocrossreact with mini-messenger RNAs containing stop signals in which U was replaced by 4-thiouridine (s4U). Here, using the same strategy we have monitored photocrosslinking to eukaryotic ribosomal components of 14-mer mRNA in the presence of tRNA(f)(Met), and 42-mer mRNA in the presence of tRNA(Asp) (tRNA(Asp) gene transcript). We show that: (a) both 14-mer and 42-mer mRNAs crossreact with ribosomal RNA and ribosomal proteins. The patterns of the crosslinked ribosomal proteins are similar with both mRNAs and sensitive to ionic conditions; (b) the crosslinking patterns obtained with 42-mer mRNAs show characteristic modification upon addition of tRNA(Asp) providing evidence for appropriate mRNA phasing onto the ribosome. Similar changes are not detected with the 14-mer mRNA.tRNA(f)(Met) pairs; (c) when eukaryotic polypeptide chain release factor 1 (eRF1) is added to the ribosome.tRNA(Asp) complex it crossreacts with the 42-mer mRNA containing the s(4)UGA stop codon located in the A site, but not with the s(4)UCA sense codon; this crosslink involves the N-terminal and middle domains of eRF1 but not the C domain which interacts with eukaryotic polypeptide chain release factor 3 (eRF3); (d) addition of eRF3 has no effect on the yield of eRF1-42-mer mRNA crosslinking and eRF3 does not crossreact with 42-mer mRNA. These experiments delineate the in vitro conditions allowing optimal phasing of mRNA on the eukaryotic ribosome and demonstrate a direct and specific contact of 'core' eRF1 and s(4)UGA stop codon within the ribosomal A site.
Chavatte
L
L
Institut Jacques Monod, UMR 7592 CNRS-Universités Paris 7-Paris 6, France.
Frolova
L
L
Kisselev
L
L
Favre
A
A
D000818:Animals; D018388:Codon, Terminator; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D006801:Humans; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D012349:RNA, Transfer, Asp / Q000737:chemistry; D012358:RNA, Transfer, Met / Q000737:chemistry; D011817:Rabbits; D012270:Ribosomes / Q000737:chemistry*; D012492:Salts / Q000494:pharmacology
D018388:Codon, Terminator; D003432:Cross-Linking Reagents; C489002:ETF1 protein, human; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D012333:RNA, Messenger; D012349:RNA, Transfer, Asp; D012358:RNA, Transfer, Met; D012492:Salts; C489006:prfB protein, E coli; D008274:Magnesium