Escherichia coli tRNA(Phe) transcript lacking all the modified nucleosides was investigated in an in vitro translation system. To estimate the affinity of tRNA toward EF-Tu, Kd and K-1 were measured by the nuclease protection assay, and it was shown that the absence of modifications decreases ternary complex stability less than 2-fold. The activity of unmodified Phe-tRNA(Phe) on E. coli ribosomes was compared to modified Phe-tRNA(Phe) using the framework of the kinetic proofreading mechanism (Thompson & Dix, 1982) with both cognate and noncognate codons. Values of the individual rate constants in the elongation process showed that the modifications increased the accuracy of translation by (1) decreasing the rate of dipeptide synthesis and (2) increasing the rate of rejection with noncognate codons.

An RNase protection assay was used to show that the dissociation rate constants and equilibrium constants of unmodified yeast and Escherichia coli phenylalanyl‐tRNA(Phes) to elongation factor Tu from E.coli were very similar to each other and to their fully modified counterparts. The affinity of aminoacylated tRNA to elongation factor Tu was substantially lower when GTP analogues were used in place of GTP, emphasizing the importance of the beta‐gamma phosphate linkage in the function of G‐proteins. Fourteen different mutations in conserved and semi‐conserved nucleotides of yeast phenylalanyl‐tRNA(Phe) were tested for binding to elongation factor Tu.GTP and assayed for activity in the ribosomal A‐ and P‐sites. Most of the mutations did not severely impair the function of these tRNAs in any of the assays. This suggests that the translational machinery does not form sequence‐specific interactions with the conserved nucleotides of tRNA.