Prokaryotic gene expression in vitro: transcription-translation coupled systems

Abstract

Transcription-translation coupled systems have been developed to study prokaryotic gene expression. Several types of expression system have been described. The original system consists of a crude unfractionated Escherichia coli extract, which supports protein synthesis directed by a template DNA. Control of gene expression at the transcriptional stage has been studied using this unfractionated system. In this respect, two examples of particular interest, lactose and tryptophan operons, are described. Other systems are either partially reconstituted or highly defined, containing up to 30 purified factors necessary for transcription (RNA polymerase) and translation (aminoacyl-tRNA synthetases, initiation, elongation and release factors). Additional differences between the various systems relate to the analysis of the gene products. Whereas most methods involve analysis of the totally synthesized protein, a particular system implies the formation of only the N-terminal di- or tripeptide of the gene product. Reconstituted systems have proved useful in studies on transcriptional, e.g., discovery and role of L factor, as well as translational regulation of gene expression, e.g., autogenous control of ribosomal protein synthesis.