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.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001435:Bacteriophages / Q000201:enzymology*; D004247:DNA; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology*; D011714:Pyrans / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D012367:RNA, Viral / Q000096:biosynthesis; D012293:Rifampin / Q000494:pharmacology; D013698:Templates, Genetic
D000900:Anti-Bacterial Agents; D011714:Pyrans; D011760:Pyrrolidinones; D012367:RNA, Viral; D000255:Adenosine Triphosphate; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium; D012293:Rifampin