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A new assay for tRNA aminoacylation kinetics

Published online by Cambridge University Press:  01 August 1998

ALEXEY D. WOLFSON ,
JEFFREY A. PLEISS  and
OLKE C. UHLENBECK
ALEXEY D. WOLFSON
Affiliation:
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA
JEFFREY A. PLEISS
Affiliation:
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA
OLKE C. UHLENBECK
Affiliation:
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA
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Abstract

An improved quantitative assay for tRNA aminoacylation is presented based on charging of a nicked tRNA followed by separation of an aminoacylated 3′-fragment on an acidic denaturing polyacrylamide gel. Kinetic parameters of tRNA aminoacylation by Escherichia coli AlaRS obtained by the new method are in excellent agreement with those measured by the conventional method. This assay provides several advantages over the traditional methods of measuring tRNA aminoacylation: (1) the fraction of aminoacyl-tRNA is measured directly; (2) data can be obtained at saturating amino acid concentrations; and (3) the assay is significantly more sensitive.

Keywords

acid gel electrophoresisalanineaminoacyl-tRNA synthetasetRNA, kinetics
Type
METHODS REPORT
Information
RNA , Volume 4 , Issue 8 , August 1998 , pp. 1019 - 1023
Copyright
© 1998 RNA Society

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