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

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A critical evolved property of all cells is their ability to sense and respond to environmental change. This is especially true of bacteria that often have to live in inhospitable and fluctuating environments. An active yet still poorly understood area of research is how bacteria sense environmental change and the mechanisms they deploy to respond to such changes. This proposal focuses on these questions in the soil-living organism Streptomyces coelicolor, which is an ideal model for two reasons. First, its genome sequence shows the organism is well armed with genes that encode proteins likely to be involved in responding to environmental stresses but the mode of action of these have yet to be described. Second, the genus Streptomyces is the source of a multitude of commercially important antibiotics, anticancer agents and immunosuppressants, the production of which are likely linked to the organism's ability to respond to environmental changes and so by understanding the underlying mechanisms we may be able to affect the production of such therapeutic molecules. Our proposal focuses on one specific group of stress sensors which are in fact complexes of two proteins: One is a sigma factor that directs the main cellular enzyme responsible for the production of specific RNA molecules (RNA polymerase) to produce proteins which allow the cell to respond to environmental change; the other is an anti-sigma factor that binds to the sigma factor and blocks its ability to bind RNA polymerase. It is the anti-sigma factor's job to sense the environmental stress. It is known that this sensing mechanism involves the disabling of the anti-sigma factor, which releases the sigma factor to coordinate the cellular response to the stress. Although it has been over 15 years since we first described the presence of what is now recognised as a widespread group of ExtraCytoplasmic Function (ECF) sigma factors (Streptomyces alone has over 50 of them encoded in its genome), we still know surprisingly little about how anti-sigma factors bind ECF sigma factors or how environmental stresses disable them. We have recently uncovered the mechanism by which Streptomyces responds to a particular form of oxidative stress (the main causative agent of ageing) known as disulfide stress. Disulfides are covalent bonds formed between the sulfur atoms of two cysteine amino acids, which are ordinarily found in proteins that get secreted from cells (e.g. hormones such as insulin) to help stabilise them in the harsh extracellular environment. Such bonds however are toxic for proteins inside the crowded environment of the cell cytoplasm where they can cause the inactivation of enzymes and the aggregation of proteins. Streptomyces responds to the appearance of intracellular disulfide bonds by inactivating a specific anti-sigma factor (RsrA), which releases its sigma factor, sigma R, to mount an anti-oxidative response. We have determined the three dimensional structure of RsrA in its resting state, i.e. before it binds sigma factor. Comparison to related protein complexes reveals that RsrA engages in a new form of molecular recognition in which the protein essentially turns itself inside-out to bind sigma R. We have also determined the structure of the deactivated form of RsrA in which an internal disulfide blocks the ability of the protein to turn inside-out. This proposal aims to capitalise on these novel observations. We will investigate how RsrA turns inside-out to bind its sigma factor. We rationalise that this mechanism could be the basis for other forms of environmental stress sensing by this large group of cellular regulators. We will therefore uncover the activation signals for a select few anti-sigma factor/sigma factor pairings, which have yet to be studied, and compare them to the RsrA/sigma R complex. Our goal is to determine if the mechanism we have discovered represents a new paradigm in environmental stress sensing in microbes.