RNA polymerase from Rhizobium japonicum.

Abstract

DNA-dependent RNA polymerase (EC 2.7.7.6) from Rhizobium japonicum was purified. The subunit structure was found to be beta beta' alpha 2 alpha, with the following apparent molecular weights determined by electrophoresis: Mr (beta and beta') 150,000 each, Mr (sigma) 96,000, Mr (alpha) 40,000, Mr (holoenzyme) 490,000, Mr (core enzyme) 380,000. The recovery of sigma was 28%. RNA polymerase from aerobically grown R. japonicum cells and from nitrogen-fixing cells have the same electrophoretic properties suggesting that no chemical modification of the enzyme occurs when cells undergo this metabolic differentiation. The enzyme is Mg2+-dependent, rifampicin-sensitive, and has optimal activity at alkaline pH (8--10) and at 35--40 degrees C. It binds strongly to bacteriophage T7 promoters, weakly to antibiotic resistance genes, and not at all to cloned R. japonicum nif DNA. Preliminary in vitro transcription experiments, including nif DNA as template, revealed that additional factors may be required for selective transcription from promoters.