In vitro experimental system for analysis of transcription-translation coupling.
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.
Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Baddiley-Clark Building, Richardson Road, Newcastle upon Tyne, NE2 4AX, UK.
Daniel
D
Castro-Roa
Nikolay
N
Zenkin
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012269:Ribosomal Proteins / Q000302:isolation & purification / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic
D012269:Ribosomal Proteins; D012321:DNA-Directed RNA Polymerases