Open AccessThe 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
Author(s) -
Berg Laila,
Lale Rahmi,
Bakke Ingrid,
Burroughs Nigel,
Valla Svein
Publication year - 2009
Publication title -
microbial biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.287
H-Index - 74
ISSN - 1751-7915
DOI - 10.1111/j.1751-7915.2009.00107.x
Subject(s) - biology , untranslated region , transcription (linguistics) , gene , five prime untranslated region , microbiology and biotechnology , promoter , mutant , messenger rna , three prime untranslated region , translational efficiency , gene expression , genetics , translation (biology) , linguistics , philosophy
Summary 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, σ 32 /σ 38 ‐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 ampicillin‐resistance 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 σ 70 ‐dependent promoter ( P1 / P anti‐tet ), demonstrating that the UTR at the DNA or RNA level has a hitherto unrecognized role in transcription.