Functional importance of the 3'-terminal adenosine of tRNA in ribosomal translation.
The universally conserved 3'-terminal CCA sequence of tRNA interacts with large ribosomal subunit RNA during translation. The functional importance of the interaction between the 3'-terminal nucleotide of tRNA and the ribosome was studied in vitro using mutant in vitro transcribed tRNA(Val) A76G. Val-tRNA(CCG) does not support polypeptide synthesis on poly(GUA) as a message. However, in a co-translation system, where Val-tRNA(CCG) represented only a small fraction of total Val-tRNA, the mutant tRNA is able to transfer valine into a polypeptide chain, albeit at a reduced level. The A76G mutation does not affect binding of Val- or NAcVal-tRNA(CCG) to the A- or P-sites as shown by efficient peptide bond formation, although the donor activity of the mutant NAcVal-tRNA(CCG) in the peptidyl transfer reaction is slightly reduced compared with wild-type NAcVal-tRNA. Translocation of 3'-CCG-tRNA from the P- to the E-site is not significantly influenced. However, the A76G mutation drastically inhibits translocation of peptidyl-tRNA G(76) from the ribosomal A-site to the P-site, which apparently explains its failure to support cell-free protein synthesis. Our results indicate that the identity of the 3'-terminal nucleotide of tRNA is critical for tRNA movement in the ribosome.
Virumae
Kai
K
Institute of Molecular and Cell Biology, Tartu University, Riia 23, Tartu 51010, Estonia.
Saarma
Urmas
U
Horowitz
Jack
J
Remme
Jaanus
J
D001483:Base Sequence; D014644:Genetic Variation; D007700:Kinetics; D016296:Mutagenesis; D017354:Point Mutation; D011068:Poly G / Q000096:biosynthesis; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000737:chemistry*; D012366:RNA, Transfer, Val / Q000235:genetics*; D013379:Substrate Specificity
D012366:RNA, Transfer, Val; D011068:Poly G; D012343:RNA, Transfer