Transcriptional Specificity of RNA Polymerase

Johnson, Ronald

Abstract

There are a variety of molecular mechanisms by which organisms can regulate gene expression during growth and development. One such mechanism is observed in B. subtilis during the process of sporulation. In response to nutrient limitations a well-defined developmental program is elicited. This developmental pathway which culminates in the dormant cell type known as an endospore is controlled, in part, by regulatory proteins (sigma factors) that alter the transcriptional specificity of RNA polymerase. The core RNA polymerase can interact with at least four different sigma factors each of which confers distinct transcriptional specificity. The central issue which we intend to address concerns the mechanism by which the sigma factors alter the template specificity of DNA-dependent RNA polymerase. In one model it has been postulated that each sigma factor induces a unique conformation in the core RNA polymerase, thereby allowing it to preferentially interact with a particular canonical promotor sequence. We plan to test this hypothesis by introducing chromophores into core RNA polymerase and spectroscopically monitoring putative structural altervations that may occur in the care enzyme when each individual sigma factor binds. Our approach involves replacing the endogenous, spectroscopically inert ZN(II) in the Beta' subunit of RNA polymerase (oligomeric structrure: Alpha2 Beta Beta'Sigma) with either Ni(II) or Co(II), thereby introducing chromophores at the metal binding sites. Similar metal ion substitutions in other systems such as E. coli RNA polymerase have been shown to be innocuous. To establish this for our system, we will examine the enzymic and physical properties of the metal-substituted derivatives. Next we will use spectroscopie techniques such as circular dichroism and difference spectorscopy to monitor putative conformational changes, upon ligand binding, in the subunit of RNA polymerase which has been shown to extensively interact with the DNA template. Similar techniques have been used to monitor ligand-induced conformational changes in E. coli Co(II)-RNA polymerase. Thus, with this approach, we should be able to correlate the transcriptional specificity of each sigma factor with its effect on the conformation of the core enzyme. These results will have important implications in regard to the mechanisms of gene regulation which may be operative in mammalian systems.