The first position of a codon placed in the A site of the human 80S ribosome contacts nucleotide C1696 of the 18S rRNA as well as proteins S2, S3, S3a, S30, and S15.
Messenger RNA analogues (42-mers) containing a GAC codon (aspartic acid) in the middle of their sequence followed by a s(4)UGA stop codon were used to identify the components of the human ribosomal A site in direct contact with the photoactivatable 4-thiouridine (s(4)U) residue. We compared the behavior of the nonphased ribosome-mRNA complex, (-)tRNA(Asp), to the one of the phased complex, (+)tRNA(Asp), in the absence and in the presence of eRF1, the eukaryotic class 1 translation termination factor of human origin. The patterns of cross-links obtained for the three complexes were similar to those previously reported for rabbit ribosomes [Chavatte, L., et al. (2001) Eur. J. Biochem. 268, 2896-2904]. Cross-links involving proteins S2, S3, S3a, and S30 were poorly dependent on the presence of tRNA(Asp) and eRF1. Cross-linking to nucleotide C1696 of 18S rRNA occurred in all complexes, but its yield was at least two times higher in the phased complex with an empty A site than in the nonphased complex or when the A site was occupied by eRF1. In contrast, protein S15 cross-linked only in the phased complex in the absence of eRF1. The data obtained point to notable differences in organization of the decoding site between mammalian and prokaryotic ribosomes and to large internal mobility of the components of the tRNA (eRF1)-free A site.
Bulygin
Konstantin
K
Institut Jacques Monod, UMR 7592 CNRS-Universites Paris 7-Paris 6, 2 place Jussieu Tour 43, 75251 Paris Cedex 05, France.
Chavatte
Laurent
L
Frolova
Ludmila
L
Karpova
Galina
G
Favre
Alain
A
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003062:Codon / Q000737:chemistry / Q000235:genetics*; D003432:Cross-Linking Reagents / Q000737:chemistry; D003596:Cytosine / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry; D012337:RNA, Ribosomal, 18S / Q000737:chemistry* / Q000235:genetics; D012349:RNA, Transfer, Asp / Q000737:chemistry / Q000235:genetics; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics*; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics; D013698:Templates, Genetic; D013891:Thiouridine / Q000737:chemistry
D003062:Codon; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D012349:RNA, Transfer, Asp; C031016:RPS3A protein, human; D012269:Ribosomal Proteins; C036004:ribosomal protein S15; C030004:ribosomal protein S2; C044152:ribosomal protein S3; C040469:ribosomal protein S30; D013891:Thiouridine; D003596:Cytosine