An improved method for the reconstitution of DNA-dependent RNA polymerase from its isolated subunits in the absence of urea is described. It allows the kinetic analysis of the assembly mechanism. The rate of reconstitution as well as the final yield are strongly temperature-dependent, whereas concentration has only a small effect. The kinetics of the formation of an intermediary complex a2p and an inactive enzyme complex a2pP'o were followed. The rate-limiting and temperature-sensitive step during reconstitution is the conversion of the inactive complex a,pp'o into active enzyme.DNA-dependent RNA polymerase from Escherichiu coli [l -31, and probably from all prokaryotes [4-121, is a multi-subunit enzyme with a subunit composition corresponding to the formula (/?'pa,) o for the monomeric enzyme particle. Little is known about the roles that the different subunits play in the complex process of transcription [2,3,13-151. The recovery of active enzyme after mixing together isolated subunits and the construction of incomplete and mixed enzyme particles enables a study of the structure, function, and genetics of RNA polymerase and its subunits.Reconstitution of active enzyme was first obtained using subunits isolated by gradient centrifugation but the separation into subunits was incomplete [17,18]. Lill and Hartmann then demonstrated that RNA polymerase inactivated by dissociation in 6 M urea was reactivated upon removal of urea by dialysis against a defined reconstitution buffer [39]. This was the basis for the first complete reconstruction of active enzyme from a stoichiometric mixture of pure subunits separated by electrophoresis on cellulose acetate strips in buffers containing 6 M urea [14,15]. Reconstitution of active enzyme was also achieved by Ishihama [20,21] Etizyme. DNA-dependent RNA polymerase (EC 2.7.7.6).
_~_The original reconstitution procedure of Heil and Zillig consisted of mixing stoichiometric amounts of the subunits suspended in a buffer containing 6 M urea, followed by dialysis of the mixture against a reconstitution buffer. Thus, it was not possible to follow the kinetics of the subunit assembly. In this paper we describe an improved method for the reconstitution of RNA polymerase in the absence of urea, and its application in a kinetic analysis of the mechanism of assembly of RNA polymerase from its subunits.
MATERIALS AND METHODSRNA polymerase from E. coli AB 301 was prepared as described earlier [24]. Rifamycin-resistant polymerase was prepared from E. coli AJ-7 1161. Core enzyme and subunit o were prepared according to Burgess [l].Separation of subunits from core enzyme was carried out by electrophoresis for 3 h at 1000 V on cellulose acetate sheets (Chemetron, Milano) in a moist chamber (H. Holzel, Dorfen) on a cooled surface, using electrophoresis buffer A (0.15 M ammonium bicarbonate pH 9.0, 0.01 M magnesium acetate, 0.02 M 2-mercaptoethanol, 1 mM EDTA, 6 M urea and 5 % glycerol). Before their electrophoresis, protein solutions were extensively dialysed against the low-salt buffer (0....
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