Preparations and properties of ribonucleic acid polymerase from Acinetobacter calcoaceticus

Abstract

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 a and # subunits appeared to be similar. No subunit corresponding to that of a from E. coli was found, and furthermore no separation between the ,B 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 A = 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.

Class 1 integrons in Acinetobacter baumannii: a weak expression of gene cassettes to counterbalance the lack of LexA-driven integrase repression

“…The A. baumannii RNA polymerase has a similar structure [30] and 50 to 70% amino acid sequence homology when compared to its E. coli counterpart, with good conservation of functional regions [23]. Despite this, the two RNA polymerases behave differently in vitro, the A. baumannii enzyme being less efficient than its E. coli counterpart [30]. Moreover, the A. baumannii and E. coli transcriptional σ 70 factor amino acid sequences are very similar, with good conservation of functional regions [23,31].…”