Use of T7 RNA polymerase in an optimized Escherichia coli coupled in vitro transcription-translation system. Application in regulatory studies and expression of long transcription units.
An Escherichia coli coupled in vitro transcription-translation system has been modified to allow efficient expression of genes under the control of a T7 promoter. We describe both the characterization and use of two S30 crude extracts prepared from E. coli, namely S30 BL21(DE3) (containing endogenous T7 RNA polymerase) and S30 BL21 (supplemented with exogenous T7 RNA polymerase). Since transcription by the highly active T7 RNA polymerase is known to overload the translational machinery of E. coli, the ratio between mRNA and ribosomes has to be regulated in the coupled in vitro system. For this purpose, the level of mRNA is controlled by varying the amount of DNA template (S30 extract with endogenous T7 RNA polymerase) or by limited amounts of exogenously added T7 RNA polymerase. The coupled in vitro system described in this paper provides two especially useful applications. First, it is most suitable for studying the regulation of gene expression in vitro, second, it can be used to express DNA templates carrying up to 10 genes. We show that genes which are not well expressed in E. coli in vivo because of unfavourable codon usage or plasmid instability are synthesized efficiently in the coupled in vitro system.
Köhrer
C
C
Institute of Medical Chemistry and Biochemistry, University of Innsbruck, Austria.
Mayer
C
C
Gröbner
P
P
Piendl
W
W
D001426:Bacterial Proteins / Q000096:biosynthesis; D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D002474:Cell-Free System; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005815:Genetic Code; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases