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

doi:10.1021/bi00081a003

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

Molecular Biosciences

Research output: Contribution to journal › Article › peer-review

82 Scopus citations

Abstract

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, K_d 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.

Original language	English (US)
Pages (from-to)	7617-7622
Number of pages	6
Journal	Biochemistry
Volume	32
Issue number	30
DOIs	https://doi.org/10.1021/bi00081a003
State	Published - 1993

ASJC Scopus subject areas

Biochemistry

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title = "In Vitro Analysis of Translational Rate and Accuracy with an Unmodified tRNA",

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.",

author = "Harrington, {K. M.} and Nazarenko, {I. A.} and Dix, {D. B.} and Thompson, {R. C.} and Uhlenbeck, {O. C.}",

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T1 - In Vitro Analysis of Translational Rate and Accuracy with an Unmodified tRNA

AU - Harrington, K. M.

AU - Nazarenko, I. A.

AU - Dix, D. B.

AU - Thompson, R. C.

AU - Uhlenbeck, O. C.

PY - 1993

Y1 - 1993

N2 - 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.

AB - 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.

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

Abstract

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