In Vitro Analysis of Translational Rate and Accuracy with an Unmodified tRNA

K. M. Harrington, I. A. Nazarenko, D. B. Dix, R. C. Thompson, O. C. Uhlenbeck*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

69 Scopus citations

Abstract

Escherichia coli tRNAPhe 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-tRNAPhe on E. coli ribosomes was compared to modified Phe-tRNAPhe 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.

Original languageEnglish (US)
Pages (from-to)7617-7622
Number of pages6
JournalBiochemistry
Volume32
Issue number30
DOIs
StatePublished - 1993

ASJC Scopus subject areas

  • Biochemistry

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