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‐Ψ‐C which is common to loop IV of practically all tRNAs used in the elongation cycle of protein synthesis reads G‐U‐Ψ‐C in tile 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 10000 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 suggest that the presence of rT in tRNA improves the fidelity of the decoding process at the A‐site of the ribosome.

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