1. The Rhodobacter capsulatus hupSLC promoter: identification of cis ‐regulatory elements and of trans ‐activating factors involved in H 2 activation of hupSLC transcription
- Author
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Bertrand Toussaint, Sylvie Elsen, Yannick Lazzaroni, Wanda Dischert, Annette Colbeau, Régis De Sury d'Aspremont, Paulette M. Vignais, Ina Delic‐Attree, and Véronique Berchet
- Subjects
Rhodobacter ,biology ,Operon ,biology.organism_classification ,Microbiology ,Molecular biology ,Primer extension ,Response regulator ,Start codon ,Regulatory sequence ,Transcription (biology) ,bacteria ,Molecular Biology ,Palindromic sequence - Abstract
Summary The [NiFe]hydrogenase of the photosynthetic bacterium Rhodobacter capsulatus is encoded by the structural hupSLC operon, the expression of which is induced by H 2 .H 2activation was no longer observable in chromosomal hupR mutants, an indication that HupR is implicated directly in the activation by H2 of hupS gene expression. The transcriptional start site of the hupS promoter, determined by primer extension mapping, was located 55 nucleotides upstream from the translational start codon of the hupS gene. Regulatory sequences were identified by serial 58 deletions of the 300 bp hupS promoter-regulatory region (phupS )a nd p hupS‐lacZ translational fusions. Cis-regulatory sequences capable of interacting with two transcription factors, IHF and HupR, a response regulator of the NtrC subfamily, were studied by electrophoretic mobility shift assays (EMSAs). The R. capsulatus IHF and HupR proteins were overexpressed in Escherichia coli and purified by affinity chromatography. IHF binds to a site, 58-TCACACACCATTG, centred at π87 nt from the transcription start site. The HupR protein binds to one site within the π162 to π152 nt region, which contains the palindromic sequence 58-TTG-R5-CAA. By the use of 58 deletions and site-directed mutagenesis of the π162/π152 region, this palindrome was shown to be required for in vivo hupS transcriptional activation by H2.
- Published
- 1997