IN-VITRO ANALYSIS OF TRANSLATIONAL RATE AND ACCURACY WITH AN UNMODIFIED TRANSFER-RNA

Escherichia coli tRNA(Phe) transcript lacking all the modified nucleos ides was investigated in an in vitro translation system. To estimate t he affinity of tRNA toward EF-Tu, K(d) and k-1 were measured by the nu clease protection assay, and it was shown that the absence of modifica tions decreases ternary complex stability less than 2-fold. The activi ty of unmodified Phe-tRNA(Phe) on E. coli ribosomes was compared to mo dified Phe-tRNA(Phe) using the framework of the kinetic proofreading m echanism (Thompson & Dix, 1982) with both cognate and noncognate codon s. Values of the individual rate constants in the elongation process s howed 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.