RESUMO
The environment of tRNA 3'-terminus in the 80S ribosomal A and P sites was studied with a tRNA(Asp) analogue that bears a 4-thiouridine residue (s4U) attached to the 3'-terminal adenosine. The tRNA(Asp) analogue was obtained by in vitro T7 transcription followed by crosslinking with [32P]ps4Up and removal of the 3'-terminal phosphate. It was shown that the presence of the additional nucleotide at the 3'-end does not to hinder the 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 crosslinking of the analogue exclusively to 28S rRNA. The crosslinking was completely dependent on the presence of s4U in the tRNA analogue. Using hydrolysis of the crosslinked 28S rRNA with RNase H in the presence of deoxyoligomers complementary to various rRNA sequences, we determined that the crosslinking occurred in fragment 4302-4540 of the 28S rRNA. This fragment is evolutionarily conservative and belongs to domain V that is involved in the formation of the peptidyl transferase site in prokaryotic ribosomes. The use of reverse transcription allowed the determination of the tRNA analogue crosslinking in the P site to nucleotides U4461 and U4502, and the analogue in the A site, to nucleotides U4469 and C4507. In addition, nucleotide C4462 was crosslinked to both P site and A site-bound tRNA analogue. An analysis of the results demonstrates that environments of the tRNA 3'-termini are closely similar in both prokaryotic and eukaryotic ribosomes.
