Molecular mechanism of environmental stress sensing by bacterial Zinc-containing Anti-Sigma factors

Lead Research Organisation: John Innes Centre
Department Name: Contracts Office

Abstract

The objectives of our proposal will be addressed through a multidisciplinary programme incorporating molecular genetics, functional genomics, biochemistry, biophysics and structural biology.
Objective i (JIC/York)- Uncovering the activation mechanisms for novel ZAS protein/sigma factor pairings in S. coelicolor. We will focus on four cytoplasmic ZAS protein/sigma factor complexes that have yet to be characterised and for which activation signals are unknown. By analogy with our past work on RsrA, we will identify these signals by first identifying the regulons for the complexes (through the creation of null mutants, ChIp-on-chip analysis & microarray transcriptional profiling, qRT-PCR). This will provide clues of the activating signals (e.g. oxidative stress responses), which will be tested on reporter constructs in vivo and on isolated (overexpressed, purified) proteins in vitro, the latter provided to York for biochemical and biophysical analysis to test whether IOPPR is the basis for signal perception. JIC will also characterise in vivo responses of RsrA mutants supplied by York that are potentially compromised in signal sensing.
Objective ii (York/JIC)- The structure of RsrARed-Zn bound to sigma R from S. coelicolor or a closely related organism determined either by X-ray crystallography or NMR. Crystallization screens of newly produced ZAS/sigma factor complexes from JIC.
Objective iii (York/JIC)- The mechanistic basis for IOPPR in the RsrARed-Zn/sigma R complex. This will be accomplished through kinetic and thermodynamic investigations of the complex where we will investigate the role of RsrARed-Zn conformational dynamics by NMR and single molecule methods and probe the relationship between fold and function of RsrA residues, especially hydrophobic residues that contribute both to the protein hydrophobic core and the interface with sigma R. These studies will yield mutants that will be supplied to JIC for in vivo testing of disulfide stress responses.

Publications

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