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