The expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA-region corresponding to the 5′-untranslated part of mRNA

Abstract

Secondary structures and the short Shine-Dalgarno sequence in the 5′-untranslated region of bacterial mRNAs (UTR) are known to affect gene expression at the level of translation. Here we report the use of random combinatorial DNA sequence libraries to study UTR function, applying the strong, σ32/σ38-dependent, and positively regulated Pm promoter as a model. All mutations in the libraries are located at least 8 bp downstream of the transcriptional start site. The libraries were screened using the ampicillinresistance gene (bla) as reporter, allowing easy identification of UTR mutants that display high levels of expression (up to 20-fold increase relative to the wild-type at the protein level). Studies of the two UTR mutants identified by a modified screening procedure showed that their expression is stimulated to a similar extent at both the transcript and protein product levels. For one such mutant a model analysis of the transcription kinetics showed significant evidence of a difference in the transcription rate (about 18-fold higher than the wild type), while there was no evidence of a difference in transcript stability. The two UTR sequences also stimulated expression from a constitutive σ70-dependent promoter (P1/Panti-tet), demonstrating that the UTR at the DNA or RNA level has a hitherto unrecognized role in transcription. © 2009 The Authors.