In vitro experimental system for analysis of transcription-translation coupling

Nucleic Acids Res. 2012 Mar;40(6):e45. doi: 10.1093/nar/gkr1262. Epub 2011 Dec 30.

Abstract

Transcription and translation are coupled in bacteria, meaning that translation takes place co-transcriptionally. During transcription-translation, both machineries mutually affect each others' functions, which is important for regulation of gene expression. Analysis of interactions between RNA polymerase (RNAP) and the ribosome, however, are limited due to the lack of an in vitro experimental system. Here, we report the development of an in vitro transcription coupled to translation system assembled from purified components. The system allows controlled stepwise transcription and simultaneous stepwise translation of the nascent RNA, and permits investigation of the interactions of RNAP with the ribosome, as well as the effects of translation on transcription and transcription on translation. As an example of usage of this experimental system, we uncover complex effects of transcription-translation coupling on pausing of transcription.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA-Directed RNA Polymerases / metabolism
  • Promoter Regions, Genetic
  • Protein Biosynthesis*
  • Ribosomal Proteins / isolation & purification
  • Ribosomal Proteins / metabolism
  • Ribosomes / metabolism
  • Templates, Genetic
  • Transcription, Genetic*

Substances

  • Ribosomal Proteins
  • DNA-Directed RNA Polymerases