Purification and characterization of bacteriophage gh-I-induced deoxyribonucleic acid-dependent ribonucleic acid polymerase from Pseudomonas putida.
Infection of Pseudomonas putida by the bacteriophage gh-L-induced the synthesis of a novel DNA-dependent RNA polymerase. This gh-L-induced RNA polymerase was purified to near homogeneity. It was shown to be distinct from the host RNA polymerase (alpha-2 beta beta sigma) physically and in respect to many of its catalytic properties. The gh-L-induced RNA polymerase was composed of a single polypeptide of approximately 98,000 molecular weight. The divalent metal ion requirement for in vitro RNA synthesis by the gh-L-polymerase could be satisified with Mg-2+, but not with Mn-2+. Rna synthesis by the gh-L polymerase was highly resistant to inhibition by rifampicin and streptolydigin but could be inhibited by relatively low concentrations of KCl or the rifamycin derivative AF/013. The structural analog of ATP, 3'-deoxyadenosine 5'-triphosphate, inhibited both the gh-L-induced and the host RNA polymerases by competing for a single binding site with ATP. The phage polymerase was extremely sensitive to this inhibitor, exhibiting an apparent K-i value (2 times 10-8 M) approximately 100 times lower than that for the host RNA polymerase. The gh-L polymerase had a highly specific template requirement for DNA from the homologous gh-L phage. It would not efficiently utilize denatured DNA templates and had only low levels of activity with pyrimidine-containing polydeoxyribonucleotide homopolymers.[1]References
- Purification and characterization of bacteriophage gh-I-induced deoxyribonucleic acid-dependent ribonucleic acid polymerase from Pseudomonas putida. Towle, H.C., Jolly, J.F., Boezi, J.A. J. Biol. Chem. (1975) [Pubmed]
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