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Development of a tRNA-dependent in vitro translation system

Published online by Cambridge University Press:  04 May 2001

RICHARD J. JACKSON ,
SAWSAN NAPTHINE  and
IAN BRIERLEY
RICHARD J. JACKSON
Affiliation:
Department of Biochemistry, University of Cambridge, Old Addenbrookes Site, Cambridge CB2 1GA, United Kingdom
SAWSAN NAPTHINE
Affiliation:
Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom
IAN BRIERLEY
Affiliation:
Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom
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Abstract

A method is described for depleting rabbit reticulocyte lysates and wheat germ extracts of endogenous tRNAs by affinity chromatography using a matrix generated by coupling ethanolamine to epoxy-activated Sepharose 6B. Greater than 90% depletion of tRNA is achieved with the result that translation becomes in effect absolutely dependent on added tRNA. This depletion procedure should prove very useful for studying the influence of tRNA concentration, and the spectrum of the tRNA population, on recoding events such as programmed frameshifting and readthrough of termination codons.

Keywords

leaky terminationprogrammed ribosomal frame-shiftingrabbit reticulocyte lysatewheat germ system
Type
Research Article
Information
RNA , Volume 7 , Issue 5 , May 2001 , pp. 765 - 773
Copyright
2001 RNA Society

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