On the role of ribosylthymine in prokaryotic tRNA function.
tRNAPhe and tRNALys were isolated from an Escherichia coli K12 mutant deficient in ribosylthymine (rT) and from the wild-type strain. The sequence G-rT-psi-C which is common to loop IV of practically all tRNAs used in the elongation cycle of protein synthesis reads G-U-psi-C in the tRNAs of the mutant strain. The purified tRNAs were compared in various steps of protein biosynthesis. The poly(U)-dependent poly(Phe) synthesis performed with purified Phe-tRNAPhe and purified elongation factors showed no dependence on the presence or absence of ribosylthymine in the respective tRNAs. In contrast, the corresponding poly(A)-dependent poly(Lys) synthesis was markedly increased when Lys-tRNALys lacking rT was used. The analysis of individual functional steps of the poly(A)-dependent elongation cycle demonstrated that the absence of rT reduced the binding to the A-site and improved the translocation reaction, whereas the formation of the ternary complex EF-Tu . GTP . aa-tRNA as well as both tRNA binding to the P-site and the peptidyltransferase reaction remained unaffected. The presence of U in place of rT in tRNA increases the misincorporation of leucine in an optimized poly(U)/poly(Phe) system from about 3 in 10 000 to 3 in 1000. Our results are in agreement with the view that rT is involved in tRNA binding to the A-site in contrast to the P-site, and suggested that the presence of rT in tRNA improves the fidelity of the decoding process at the A-site of the ribosome.
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D010441:Peptide Chain Elongation, Translational; D010444:Peptide Elongation Factor Tu; D011061:Poly A / Q000378:metabolism; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D014529:Uridine / Q000031:analogs & derivatives* / Q000172:deficiency
D001426:Bacterial Proteins; C009182:ribothymidine; D011061:Poly A; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu; D014529:Uridine