Positioning of the mRNA stop signal with respect to polypeptide chain release factors and ribosomal proteins in 80S ribosomes

FEBS Letters

Bulygin, Konstantin N²  Graifer, Dmitri M²  Repkova, Marina N²  Ven'yaminova, Aliya G²  Frolova, Ludmila Yu¹  Kisselev, Lev L¹  Karpova, Galina G² 

[1] Engelhardt Institute of Molecular Biology, the Russian Academy of Sciences, Moscow 119991, Russia;Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk 630090, Russia

关键词: Translation termination;    Human ribosome;    mRNA analog;    Photocrosslinking;    Eukaryotic release factor 1;    Eukaryotic release factor 3;   

DOI  :  10.1016/S0014-5793(02)02304-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

To study positioning of the mRNA stop signal with respect to polypeptide chain release factors (RFs) and ribosomal components within human 80S ribosomes, photoreactive mRNA analogs were applied. Derivatives of the UUCUAAA heptaribonucleotide containing the UUC codon for Phe and the stop signal UAAA, which bore a perfluoroaryl azido group at either the fourth nucleotide or the 3′-terminal phosphate, were synthesized. The UUC codon was directed to the ribosomal P site by the cognate tRNA^Phe, targeting the UAA stop codon to the A site. Mild UV irradiation of the ternary complexes consisting of the 80S ribosome, the mRNA analog and tRNA resulted in tRNA-dependent crosslinking of the mRNA analogs to the 40S ribosomal proteins and the 18S rRNA. mRNA analogs with the photoreactive group at the fourth uridine (the first base of the stop codon) crosslinked mainly to protein S15 (and much less to S2). For the 3′-modified mRNA analog, the major crosslinking target was protein S2, while protein S15 was much less crosslinked. Crosslinking of eukaryotic (e) RF1 was entirely dependent on the presence of a stop signal in the mRNA analog. eRF3 in the presence of eRF1 did not crosslink, but decreased the yield of eRF1 crosslinking. We conclude that (i) proteins S15 and S2 of the 40S ribosomal subunit are located near the A site-bound codon; (ii) eRF1 can induce spatial rearrangement of the 80S ribosome leading to movement of protein L4 of the 60S ribosomal subunit closer to the codon located at the A site; (iii) within the 80S ribosome, eRF3 in the presence of eRF1 does not contact the stop codon at the A site and is probably located mostly (if not entirely) on the 60S subunit.

【 授权许可】

Unknown