Control of Formation of RNA Polymerase in Escherichia coli

Abstract

INTRODUCTION
The DNA-dependent RNA polymerase (EC 2.7.7.6) of Escherichia coli is composed of at least four different polypeptide chains, and, so far, two forms of the holoenzyme with the subunit structures α₂ββ′ σ and α₂ββ′ σ′ have been identified (Burgess et al. 1969; Fukuda, Iwakura and Ishihama 1974). Of the two holopolymerases, only holoenzyme II possesses the activities needed to carry out the de novo synthesis of [poly(A)] · [poly(U)] from ATP and UTP in the presence of manganese ion, but without DNA, and the [poly(A)] synthesis directed by double-stranded DNA (Iwakura, Fukuda and Ishihama 1974). These differences in catalytic properties suggested that the two holopolymerases play different roles in transcription or RNA synthesis. The finding of the heterogeneity of RNA polymerase in E. coli raised the possibility that transcription is regulated, at least in part, by the control of concentration of the individual polymerase molecules. The present study is concerned with the regulation of the synthesis of individual subunits and of their subsequent assembly into the complex enzyme structures. Parts of this work have appeared elsewhere (Ishihama et al. 1976; Taketo and Ishihama 1976).

EXPERIMENTAL PROCEDURES
Chemicals
³H- and ¹⁴C-labeled amino acids were obtained from Radiochemical Centre (England) and Daiichi Purechemicals (Japan), respectively. Unlabeled and labeled ribonucleoside triphosphates were purchased from Boehringer (Germany) and Schwarz Bioresearch (U.S.A.), respectively. Synthetic deoxypolymers were products of P-L Biochemicals (U.S.A.). DNA of phages T4 and T7 was purified by phenol extraction from phage stocks, which were prepared by centrifugation in CsCl solutions. Pancreatic...