THE RHODOBACTER-CAPSULATUS HUPSLC PROMOTER - IDENTIFICATION OF CIS-REGULATORY ELEMENTS AND OF TRANSACTIVATING FACTORS INVOLVED IN H-2 ACTIVATION OF HUPSLC TRANSCRIPTION

Citation
B. Toussaint et al., THE RHODOBACTER-CAPSULATUS HUPSLC PROMOTER - IDENTIFICATION OF CIS-REGULATORY ELEMENTS AND OF TRANSACTIVATING FACTORS INVOLVED IN H-2 ACTIVATION OF HUPSLC TRANSCRIPTION, Molecular microbiology, 26(5), 1997, pp. 927-937
Citations number
43
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology,Microbiology
Journal title
ISSN journal
0950-382X
Volume
26
Issue
5
Year of publication
1997
Pages
927 - 937
Database
ISI
SICI code
0950-382X(1997)26:5<927:TRHP-I>2.0.ZU;2-W
Abstract
The [NiFe]hydrogenase of the photosynthetic bacterium Rhodobacter caps ulatus is encoded by the structural hupSLC operon, the expression of w hich is induced by H-2. H-2 activation was no longer observable in chr omosomal hupR mutants, an indication that HupR is implicated directly in the activation by H-2 of hupS gene expression. The transcriptional start site of the hupS promoter, determined by primer extension mappin g, was located 55 nucleotides upstream from the translational start co don of the hupS gene. Regulatory sequences were identified by serial 5 ' deletions of the 300 bp hupS promoter-regulatory region (phupS) and phupS-lacZ translational fusions, Cis-regulatory sequences capable of interacting with two transcription factors, IHF and HupR, a response r egulator of the NtrC subfamily, were studied by electrophoretic mobili ty shift assays (EMSAs). The R. capsulatus IHF and HupR proteins were overexpressed in Escherichia coil and purified by affinity chromatogra phy, IHF binds to a site, 5'-TCACACACCATTG, centred at -87 nt from the transcription start site, The HupR protein binds to one site within t he -162 to -152 nt region, which contains the palindromic sequence 5'- TTG-R-5-CAA. By the use of 5' deletions and site-directed mutagenesis of the -162/-152 region, this palindrome was shown to be required for in vivo hupS transcriptional activation by H-2.