Promoter activities in Corynebacterium glutamicum strains with deletions of genes encoding sigma factors of RNA polymerase suggested that transcription from some promoters is controlled by two sigma factors. To prove that different sigma factors are involved in the recognition of selected Corynebacterium glutamicum promoters, in vitro transcription system was applied. It was found that a typical housekeeping promoter Pper interacts with the alternative sigma factor σ(B) in addition to the primary sigma factor σ(A). On the other way round, the σ(B)-dependent promoter of the pqo gene that is expressed mainly in the stationary growth phase was active also with σ(A). Some promoters of genes involved in stress responses (P1clgR, P2dnaK, and P2dnaJ2) were found to be recognized by two stress-responding sigma factors, σ(H) and σ(E). In vitro transcription system thus proved to be a useful direct technique for demonstrating the overlap of different sigma factors in recognition of individual promoters in C. glutamicum.
- MeSH
- brouci fyziologie metabolismus MeSH
- celulosa MeSH
- doškolování MeSH
- lidé MeSH
- mikrobiologické jevy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Geografické názvy
- Nový Zéland MeSH
To facilitate transcription studies in Corynebacterium glutamicum, we have developed an in vitro transcription system for this bacterium used as an industrial producer of amino acids and a model organism for actinobacteria. This system consists of C. glutamicum RNA polymerase (RNAP) core (α2, β, β'), a sigma factor and a promoter-carrying DNA template, that is specifically recognized by the RNAP holoenzyme formed. The RNAP core was purified from the C. glutamicum strain with the modified rpoC gene, which produced His-tagged β' subunit. The C. glutamicum sigA and sigH genes were cloned and overexpressed using the Escherichia coli plasmid vector, and the sigma subunits σ(A) and σ(H) were purified by affinity chromatography. Using the reconstituted C. glutamicum holo-RNAPs, recognition of the σ(A)- and σ(H)-dependent promoters and synthesis of the specific transcripts was demonstrated. The developed in vitro transcription system is a novel tool that can be used to directly prove the specific recognition of a promoter by the particular σ factor(s) and to analyze transcriptional control by various regulatory proteins in C. glutamicum.
- MeSH
- Corynebacterium glutamicum genetika metabolismus MeSH
- DNA řízené RNA-polymerasy genetika metabolismus MeSH
- druhová specificita MeSH
- genetická transkripce * MeSH
- genetické techniky MeSH
- molekulární sekvence - údaje MeSH
- promotorové oblasti (genetika) * MeSH
- regulace genové exprese u bakterií * MeSH
- sekvence nukleotidů MeSH
- sigma faktor genetika metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
