Preparations and properties of ribonucleic acid polymerase from Acinetobacter calcoaceticus.
Deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase (EC 2.7.7.6) from Acinetobacter calcoaceticus was purified to apparent homogeneity and its properties were compared with those of the Escherichia coli B enzyme. The molecular weights of the two native active enzymes as well as their alpha and beta subunits appeared to be similar. No subunit corresponding to that of sigma from E. coli was found, and furthermore no separation between the beta subunits could be detected by gel electrophoresis. A number of different DNAs were transcribed by the enzyme from A. calcoaceticus. Maximal RNA synthesis occurred at pH 8.7, 10 mM Mg2+, or 0.3 mM Mn2+ and at a total ionic strength of 0.1. Higher ionic strengths led to increasing inhibition of transcription and at mu = 0.4 complete inhibition was observed. The mechanism of inhibition of salt was not related to the initiation event as observed with T4 core RNA polymerase (R.Kleppe, 1975). In an attempt to understand the mechanism of inhibition by salt, the effect of ionic strength on the sedimentation properties of the enzyme was investigated. At low ionic strength, enzyme species with sedimentation coefficients, s20,w, of 5.8S, 12.4S, and 19.3S were present. In buffers with higher ionic strengths the relative amounts of the 12.4S species decreased. It is suggested, therefore, that the inhibition of activity at higher salt concentrations is caused by a decrease in concentration of the active enzyme species.
D000150:Acinetobacter / Q000201:enzymology*; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002474:Cell-Free System; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D011189:Potassium Chloride / Q000494:pharmacology; D012329:RNA, Bacterial / Q000096:biosynthesis; D012293:Rifampin / Q000494:pharmacology; D014158:Transcription, Genetic
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D008345:Manganese; D011189:Potassium Chloride; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium; D012293:Rifampin