Downloaded from www.microbiologyresearch.org by IP: 54.144.26.64 On: Wed, 27 Jan 2016 10:56:31 Microbiology (1 998), 144,2855-2864 Printed in Great Britain Selection and characterization of mercury- independent activation mutants of the Tn501 transcriptional regulator, MerR Julian Parkhill,t Blair Lawley,* Jonathan L. Hobman and Nigel L. Brown Author for correspondence: Nigel L. Brown. Tel: +44 121 414 5467. Fax: +44 121 414 5907. e-mail : n.l.brown@bham.ac.uk Microbial Molecular Genetics and Cell Biology Research Group, School of Biological Sciences, The University of Birmingham, Edgbaston, Birmingham 615 2lT, UK MerR is the transcriptional regulator of the mercury-resistance (mer) operon of transposon TnSO1, acting at the mer promoter as both an activator in the presence of mercuric salts and a repressor in their absence. This paper reports a method for selection of constitutive activator mutants, which activate transcription in the absence of Hg", and the characterization of these MerRAC proteins. At least two mutations in the MerR protein were found necessary for strong mercury-independent activation, and these mutations lie in the C- terminal two-thirds of the MerR protein near the Hg"-binding cysteines. A triple mutation was shown to increase activation over the corresponding double mutations. All mutant proteins caused further activation in the presence of Hg". The data support a mechanism in which a conformational change of one or both MerR subunits in the homodimer drives a distortion of DNA bound t o a helix-turn-helix structure in the N-terminal region. A mutation in this putative helix-turn-helix region severely reduced both the repressor and activator functions of MerR. Keywords : repressor, activator, MerR family, transposon, mercury resistance INTRODUCTION MerR from TnSO1 regulates expression of the mercury- resistance (mer) genes. In the absence of mercury it represses transcription of the mercury-resistance operon (merTPAD) from a single promoter, P,, and in the presence of Hg" it activates transcription several hun- dredfold from the same promoter (Lund & Brown, 1989a). It also maintains repression of its own promoter, P, (Lund et al., 1986). All these functions are carried out by a single MerR homodimer bound to the dyad symmetrical operator DNA between the -35 and - 10 elements of P, (O'Halloran & Walsh, 1987). Unlike most positively controlled promoters (Raibaud & Schwartz, 1984), both the -35 and - 10 elements of P, show good similarity to those of a canonical Escherichia coli 0'' promoter (5/6 and 3/6 respectively; Lund et al., 1986). However, the constitutive promoter activity of P, is very weak, due to the abnormally long spacer t Present address: The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 lSA, UK. *Present address: School of Molecular and Medical Biosciences, Cardiff University, PO Box 920, Cardiff CF1 3XP, UK. sequence between the promoter elements (19 bp com- pared to 17_+ 1 bp in a typical promoter; Harley & Reynolds, 1987), which is essential to the normal induction of the promoter (Lund & Brown, 1989b; Parkhill & Brown, 1990). Both MerR and RNA poly- merase remain bound to the promoter during induction and some of the contacts of MerR with the DNA at the centre of the operator are altered on induction (O'Halloran et al., 1989; Frantz & O'Halloran, 1990; Heltzel et al., 1990). Mutations in the RNA polymerase 01 and 0'' subunits can affect the expression of P,, but the basis for this is not known (Caslake et al., 1997) and, unlike a number of other activators, MerR does not contact site I of the a-subunit (A. Ansari et al., unpublished data, quoted in Ishihama, 1993). These observations led to a model for P, activation in which MerR, on binding Hg", directly causes a dis- tortion of the DNA at the promoter, realigning the - 10 and -35 elements and facilitating open complex for- mation by RNA polymerase (Lund & Brown, 1989a; O'Halloran et al., 1989). O'Halloran and co-workers have shown that the addition of Hg" salts to the MerR-DNA complex caused unwinding by 33" and straightening of a MerR-induced bend in the operator ~ 0002-2644 0 1998 SGM 2855