Award details

The physiological and biochemical function of sigma-28 in Streptomyces coelicolor A3 (2)

Reference	G04257
Principal Investigator / Supervisor	Professor Mark Buttner
Co-Investigators / Co-Supervisors
Institution	University of East Anglia
Department	Biological Sciences
Funding type	Research
Value (£)	125,987
Status	Completed
Type	Research Grant
Start date	01/10/1994
End date	01/10/1997
Duration	36 months

Abstract

We have cloned the gene (sigE) encoding the 28kD RNA polymerase sigma factor, sigma28, from S. coelicolor and used the cloned DNA to generate a sigE null mutant which proved to be unable to grow on minimal medium. sigma28 lacks the amino acid sequences predicted to be responsible for masking DNA binding in the absence of the core subunits. sigE will be over-expressed in E. coli and milligram quantities of sigma28 will be purified. This material will be used (a) in in vitro transcription studies to identify new promoters (and hence genes) of the sigma28 regulon, (b) to determine if sigma28 will bind to its cognate promoter, dagAP2, in the absence of core RNA polymerase and (c) to set up pilot experiments toward a structural determination of sigma28 using high- field NMR and X-ray crystallography. The biological role of sigma28 will be explored through the investigation of the phenotype of the sigE mutant, the analysis of the consequences of sigma28 over-expression in S. coelicolor and an examination of the effects of growth phase and nutritional status on sigE expression. As a general aid to RNA polymerase work in Streptomyces, a one-step purification method for S. coelicolor RNA polymerase will be established, using an approach recently developed for the E. coli enzyme. The method is based on the his-tagging of the S. coelicolor RNA polymerase beta' subunit (rpoC) gene, which we have already isolated and partly sequenced.

Summary

unavailable

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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