Analysis of aminoacyl- and peptidyl-tRNAs by gel electrophoresis.
During protein synthesis, ribosomes translate 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 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.
Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA, USA.
Brian D
BD
Janssen
Elie J
EJ
Diner
Christopher S
CS
Hayes
D015152:Blotting, Northern; D019327:Copper Sulfate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004917:Erythromycin / Q000494:pharmacology; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D012329:RNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification*; D012346:RNA, Transfer, Amino Acyl / Q000032:analysis* / Q000302:isolation & purification* / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism
D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; C019037:tRNA, peptidyl-; D004917:Erythromycin; D019327:Copper Sulfate