Arrangement of 3'-terminus of tRNA on the human ribosome as revealed from cross-linking data.
This study is directed towards an important problem concerning the organization of the peptidyl transferase center (PTC) on the mammalian ribosome that cannot be studied by X-ray analysis since crystals of 80S ribosomes are still unavailable. Here, we investigated the arrangement of the 3'-end of tRNA in the 80S ribosomal A and P sites using a tRNA(Asp) analogue that bears a 4-thiouridine (s(4)U) attached to the 3'-terminal adenosine. It was shown that an additional nucleotide s(4)U77 on the 3'-end does not impede codon-dependent binding of the tRNA to the A and P sites of 80S ribosome. Mild UV-irradiation of the ribosomal complexes containing a short appropriately designed mRNA and the tRNA analogue resulted in cross-linking of the analogue exclusively to 28S rRNA. The cross-linking site was detected in the 4302-4540 fragment of the 28S rRNA which belongs to the highly conserved domain V that in prokaryotic ribosomes is involved in the formation of the PTC. Nucleotides cross-linked to the tRNA analogue were determined by means of reverse transcription. A comparison of the results obtained with a dynamic model of mutual arrangement of s(4)U77 of the A site tRNA and nucleotides of 23S rRNA built on the basis of an atomic model for the prokaryotic PTC led to the conclusion that environments of the tRNA 3'-terminus in prokaryotic and eukaryotic ribosomes share a significant extent of similarity, although pronounced differences are also detectable.
Bulygin
Konstantin
K
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
Favre
Alain
A
Baouz-Drahy
Soria
S
Hountondji
Codjo
C
Vorobjev
Yury
Y
Ven'yaminova
Aliya
A
Graifer
Dmitri
D
Karpova
Galina
G
D001483:Base Sequence; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000737:chemistry; D012338:RNA, Ribosomal, 23S / Q000737:chemistry; D012339:RNA, Ribosomal, 28S / Q000737:chemistry; D012349:RNA, Transfer, Asp / Q000737:chemistry*; D012360:RNA, Transfer, Phe / Q000737:chemistry*; D012270:Ribosomes / Q000378:metabolism*
D012333:RNA, Messenger; D012338:RNA, Ribosomal, 23S; D012339:RNA, Ribosomal, 28S; D012349:RNA, Transfer, Asp; D012360:RNA, Transfer, Phe