RNA polymerases (RNA Pol) from various sources are known to contain intrinsic metal(s). Procarytoic enzymes have been shown to contain 2 g-atom Zn/mol enzyme. The catalytic and structural roles of one of these metals, which is located in beta subunit of E. coli RNA Pol, have been established. The function of the other metal in beta subunit of E. coli enzyme is still not clear. In this proposed project, Bacillus subtilis RNA Pol will be used to study the role of this other intrinsic metal. This enzyme is superior to E. coli RNA Pol for the intended study because both metals are in beta subunit. More importantly, removal of these metals does not require the harsh, extreme low pH conditions, minimizing possible inactivation of the binding sites. Accordingly, intrinsic metals of B. subtilis RNA Pol will be removed by RSTA in urea. Reconstitution of the denatured, apoenzyme in the presence of externally added metal ions will be performed under appropriate pH, metal concentration, ionic strength, and temperature conditions to yield apo, half-apo, and metal-hybrid enzymes. Subunit and sub-chain localization of the reconstituted enzyme will be performed by affinity chromatography and limited proteolysis study. Purified apo-, half-apo, and metal-hybrid enzymes, with metal location now determined, will be used in selected biochemical (catalytic) and biophysical (structural) studies. Comparison of results from these studies should allow deduction of the function(s) of each intrinsic metal. Results from this project should provide valuable insights into such important biomedical issues as metal-regulated gene expression and metal- ion carcinogenesis.
