Analysis of Aminoacyl- and Peptidyl-tRNAs by Gel Electrophoresis

Abstract

During protein synthesis, the ribosome translates the genetic information encoded within messenger RNAs into defined amino acid sequences. Transfer RNAs (tRNAs) are crucial adaptor molecules in this process, delivering amino acid residues to the ribosome and holding the nascent peptide chain as it is assembled. Here, we present methods for the analysis of aminoacyl- and peptidyl-tRNA species isolated from Escherichia coli. These approaches utilize denaturing gel electrophoresis at acidic pH to preserve the labile ester bonds that link amino acids to tRNA. Specific aminoacyl- and peptidyl-tRNAs are detected by Northern blot hybridization using probes for tRNA isoacceptors. Small peptidyl-tRNAs can be differentiated from aminoacyl-tRNA through selective deacylation of the latter with copper sulfate. Additionally, peptidyl-tRNAs can be detected through metabolic labeling of the nascent peptide. This latter approach is amenable to pulse-chase analysis to examine peptidyl-tRNA turnover in vivo. We have applied these methods to study programmed translational arrests and the kinetics of paused ribosome turnover.

A Highly Adaptable Method for Quantification of Peptidyl-tRNA Hydrolase Activity

“…Based on the typical size of tRNA, acrylamide percentages from 4 to 15% were tested. An acrylamide percentage of 6.5% yielded the largest separation of substrate peptidyltRNA from product tRNA, in agreement with the optimum acrylamide percentages determined for previous tRNA separations for Northern blotting [23,24]. Minigels with higher percentages of acrylamide exhibited slower sample migration and decreased separation.…”

Reduction of translating ribosomes enables Escherichia coli to maintain elongation rates during slow growth

“…Total aminoacyl-tRNA was extracted from cells under acidic conditions and subjected to acid gel electrophoresis as described in Janssen et al 64 using tRNA-specific hybridization probes as listed in Dong et al 33 .…”

Structure and mechanism of the tRNA-dependent lantibiotic dehydratase NisB

“…To assess the extent of aminoacylation on in vitro transcribed E. coli tRNA Glu , glutamyl-tRNA Glu was analyzed by denaturing acidic polyacrylamide gel using a previously described procedure 32,33 . tRNA aminoacylation assays were performed using L-[ 14 C(U)]-glutamic acid (0.1 µCi µL −1 , 260 mCi mmol −1 ) under the following conditions: in vitro transcribed E. coli tRNA Glu (3 µM), L-[ 14 C(U)]-glutamic acid (50 µM), HEPES pH 7.5 (100 mM), ATP (5 mM), TCEP (1 mM), KCl (10 mM), MgCl 2 (20 mM), TIPP (0.02 U µL −1 ) and purified E. coli GluRS (1 µM) in a final volume of 50 µL.…”

“…To remove any aminoacylated tRNA, total RNA (0.66 µg µL −1 ) was incubated for 1 h at 37 °C in EDTA (0.6 mM) and Tris-HCl pH 9.0 (66 mM). The sample was acidified with NaOAc (300 mM) and RNA was extracted by standard EtOH precipitation 33 .…”