Subunit Composition of Escherichia coli RNA Polymerase during Transcription in vitro

Abstract

The subunit composition of E. coli RNA polymerase during in vitro transcription of bacteriophage T7DNA was analyzed at several steps of RNA synthesis. RNA-polymerase.cntdot.DNA complexes were sedimented through a glycerol gradient, and the RNA polymerase subunits present in each fraction of the gradient were separated by dodecylsulfate-polyacrylamide gel electrophoresis and quantified colorimetrically on the gels. RNA polymerase selectively bound to T7 DNA in the absence of nucleoside triphosphates has the same subunit composition as free RNA polymerase holoenzyme (.beta.''.beta..alpha.2).sigma.. Addition of the nucleoside triphosphate combinations ATP, GTP, UTP or ATP, CTP, UTP or GTP, CTP, UTP to the binding reaction does not alter the subunit composition of RNA polymerase holoenzyme bound to DNA. In the presence of ATP, GTP and CTP up to 3 pmol of .sigma.-subunit are released from a complex containing RNA polymerase and 1 pmol of T7 DNA. In the presence of the 4 nucleoside triphosphates about 90% of the RNA polymerase associated with DNA and nascent RNA has the subunit composition of RNA polymerase core enzyme (.beta.''.beta..alpha.2). The .sigma.-subunit is released from the complex and is recovered near the top of the gradient. The transition from the binding complex to the elongation complex and the incorporation of .gamma.32P-labeled ATP and GTP at the 5'' end of RNA molecules were followed in parallel. In the purified elongation complex about 1 pmol of ATP or GTP is incorporated into RNA per pmol RNA polymerase core enzyme engaged in RNA synthesis.