title: Construction of in vitro transcription system for Corynebacterium glutamicum and its use in the recognition of promoters of different classes.
abstract: 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.
authors.affiliation: Institute of Microbiology, AS CR, v.v.i., Vídeňská 1083, 14220, Prague 4, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Holátko
authors.forename: Radoslav
authors.initials: R
authors.lastname: Silar
authors.forename: Alžbeta
authors.initials: A
authors.lastname: Rabatinová
authors.forename: Hana
authors.initials: H
authors.lastname: Sanderová
authors.forename: Petr
authors.initials: P
authors.lastname: Halada
authors.forename: Jan
authors.initials: J
authors.lastname: Nešvera
authors.forename: Libor
authors.initials: L
authors.lastname: Krásný
authors.forename: Miroslav
authors.initials: M
authors.lastname: Pátek
mesh_terms: D001483:Base Sequence; D048230:Corynebacterium glutamicum / Q000235:genetics* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D014158:Transcription, Genetic
chemical_list: D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases