A new assay for tRNA aminoacylation kinetics.
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.
Wolfson
A D
AD
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Pleiss
J A
JA
Uhlenbeck
O C
OC
D000412:Alanine-tRNA Ligase / Q000378:metabolism*; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D012346:RNA, Transfer, Amino Acyl / Q000032:analysis* / Q000378:metabolism; D046249:Transfer RNA Aminoacylation
D012346:RNA, Transfer, Amino Acyl; D000412:Alanine-tRNA Ligase