The intent of the proposed research is to extend our present knowledge of the mechanism of action of RNA polymerase. The specific aims of the project are: 1) Reconstitution of RNA polymerase containing chemically modified sigma and alpha subunits in order to define the role of these subunits. 2) Peptide mapping of polymerase subunits by limited proteolysis in SDS. 3) Using appropriate precursor and product analogues and incubation conditions affinity labeling of the initiation, elongation and product sites of RNA polymerase will be attempted. 4) As a possible approach toward a characterization of the mechanism of translocation the incorporation of 32P into NTP during pyrophosphate exchange directed by templates of alternating sequence will be determined.