On the Role of Ribosylthymine in Prokaryotic tRNA Function

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Abstract
TRNAPhe and tRNALys were isolated from an Escherichia coli K12 mutant deficient in ribosylthymine (rT) and from the wild-type strain. The seqeunce G-rT-.PSI.-C which is common to loop IV of practically all tRNA used in the elongation cycle or protein synthesis reads G-U-.PSI.-C in the tRNA of the mutant strain. The purified tRNA 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 tRNA. The corresponding poly(A)-dependent poly(Lys) synthesis was markedly increased when Lys-tRNALys lacking rT was used. The analysis of indiviual 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; the formation of the ternary complex EF [elongation facotr] Tu.cntdot.GTP.cntdot.aa[aminoacyl]-tRNA the 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. Ribosylthymine is apparently involved in tRNA binding to the A-site in contrast to the P-site. The presence of rT in tRNA evidently improves the fidelity of the decoding process at the A-site of the ribosome.