Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase of Pseudomonas putida

Abstract

Abstract Two forms of RNA polymerase (nucleoside triphosphate-RNA nucleotidyltransferase, EC 2.7.7.6) from Pseudomonas putida were resolved by chromatography in 50% glycerol on phosphocellulose and purified. As determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, the subunit structures of the two forms of the enzyme were α2ββ'σ and α2ββ'. The molecular weights of α, β, β', and σ were 44,000, 155,000, 165,000, and 98,000, respectively. The sedimentation coefficients of α2ββ'σ and α2ββ' as determined by sucrose gradient centrifugation in 0.40 m potassium acetate were 13 S and 12 S, respectively, but in 0.05 m potassium acetate, the sedimentation coefficients were 19 S and 25 S. Similar results were obtained by analytical ultracentrifugation. Multiple protein bands resulted from polyacrylamide disc gel electrophoresis of α2ββ'σ and α2ββ'. Each band was enzymatically active in the unprimed synthesis of poly A · poly U. RNA polymerase α2ββ'σ was 4- to 5-fold more active in transcribing P. putida bacteriophage gh-1 DNA and coliphage T4 DNA than was α2ββ'. With calf thymus DNA as a template, the two forms were equally active in RNA synthesis. With denatured DNA or poly d(A-T), α2ββ' was twice as active as was α2ββ'σ. The rates of the DNA-directed polymerization reactions catalyzed by α2ββ'σ and α2ββ' were affected differently by added RNase. The initial rate of the reaction catalyzed by α2ββ'σ was increased 10%, whereas that catalyzed by α2ββ' was increased 80 to 110%.