RNA Polymerase Assembly In Vitro: Reactivation of Denatured Core Enzyme and Studies of Assembly Intermediates

Abstract

INTRODUCTION
A complete understanding of the processes controlling transcription and thus leading to differential gene expression requires a detailed analysis of the interrelationship of RNA polymerase structure and function. An important aspect of the investigation of this problem is the elucidation of the role of each of the RNA polymerase subunits in the overall transcription process and the subunit interactions required for the attainment of native conformation and functional capability.

To facilitate this investigation we have sought a means of denaturing and reactivating RNA polymerase that allows complete recovery of enzymatic activity and structural parameters. This has been accomplished by incubation of the E. coli RNA polymerase core molecule in 6 M guanidine hydrochloride followed by removal of the denaturant by dialysis against glycerol-containing buffer (Harding and Beychok 1974). Several other investigators have performed related studies utilizing urea denaturation (Heil and Zillig 1970; Lill and Hartmann 1970; Ishihama and Ito 1972). Reactivation of enzyme denatured by guanidine or urea is markedly temperature-dependent (Fukuda and Ishihama 1974; Harding and Beychok 1974; Yarbrough and Hurwitz 1974a; Lill, Behrendt and Hartmann 1975; Palm et al. 1975) and independent of the presence of sigma subunit (Harding and Beychok 1974; Yarbrough and Hurwitz 1974a; Lill, Behrendt and Hartmann 1975). Some physical and functional properties of renatured preparations have also been determined. We have shown (Harding and Beychok 1974 and below) that native and renatured preparations have, within experimental error, identical sedimentation coefficients. Stokes’ radii and far UV CD (circular dichroism) spectra. Yarbrough and Hurwitz (1974a)...