1887

Abstract

The KstR-dependent promoter of the gene of , which encodes the 3-β-hydroxysteroid dehydrogenase (3-β HSD) responsible for the first step in the cholesterol degradative pathway, has been characterized. Primer extension analysis of the promoter showed that the transcription starts at the ATG codon, thus generating a leaderless mRNA lacking a 5′ untranslated region (5′UTR). Footprint analyses demonstrated experimentally that KstR specifically binds to an operator region of 31 nt containing the quasi-palindromic sequence AACTGGAACGTGTTTCAGTT, located between the −5 and −35 positions with respect to the transcription start site. This region overlaps with the −10 and −35 boxes of the promoter, suggesting that KstR represses transcription by preventing the binding of RNA polymerase. Using a –β-galactosidase fusion we have demonstrated that KstR is able to work as a repressor in a heterologous system like . A 3D model of the KstR protein revealed folding typical of TetR-type regulators, with two domains, i.e. a DNA-binding N-terminal domain and a regulator-binding C-terminal domain composed of six helices with a long tunnel-shaped hydrophobic pocket that might interact with a putative highly hydrophobic inducer. The finding that similar promoter regions have been found in all mycobacterial strains examined, with the sole exception of , provides new clues about the role of cholesterol in the pathogenicity of this micro-organism.

Funding
This study was supported by the:
  • Ministry of Science and Innovation (Award BFU2009-11545-C03-03 and BFU2006-15214-C03-01)
  • Consejo Superior de Investigaciones Científicas (Award BFU2006-15214-CO3-01)
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2011-09-01
2021-05-15
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