Dna‐Dependent rna polymerase from E. coli: Structure investigation

Abstract

E. coli RNA polymerase holoenzyme consists of two large subunits--8 and p' (molecular weights 155,000 and 165,000), of two identical a subunits (molecular weight 36,500), and of the initiation factor u (molecular weight 86,000). The complete amino acid sequence of the RNA polymerase a subunit was determined. According to the primary structure the polypeptide chain of the a subunit has a molecular weight of 36,512 and consists of 329 amino acid residues. In order to determine the primary structure of large -8 and p' subunits of E. coli RNA polymerase, the protein fragmentation and sequencing procedure was combined with sequencing of the corresponding structural genes, which facilitated the ordering of the peptide fragments obtained during sequencing of the polypeptide chains. In all, 4714 base pairs of the rpoBC operon were sequenced including the entire structural gene of the -8 subunit and an initial segment (528 base pairs) of the p' structural gene. The -8 subunit appeared to consist of 1342 amino acid residues from which molecular mass was calculated to be 15061 8.6 daltons. The spatial organization of DNA-dependent RNA polymerase was studied with the aid of chemical modification and limited proteolysis. Photochemical affinity modifications were used to find out subunits of the enzyme responsible for binding of the DNA template and the RNA product.