The Reconstitution of Escherichia coli DNA-Dependent RNA Polymerase from Its Isolated Subunits

Abstract

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....

RNA Polymerase: Interaction of RNA and Rifampicin with the Subassembly α2β

“…It has been observed that a stable intermediary subassembly is formed during the genesis of RNA polymerase holoenzyme from its subunits. This subassembly consists of only two kinds of subunits with the stoichiometry x 2 B [2,3] and, like the complete enzyme, binds rifampicin [4-61. RNA, the product of transcription, can inhibit the binding of rifampicin to this subassembly [7].…”

Expression of cloned rpoB gene of Escherichia coli: a genetic system for the isolation of dominant negative mutations and overproduction of defective beta subunit of RNA polymerase

“…The advantage of the T7 overexpression system (43) is that it allows massive production of transcriptionally poisonous 1 subunit without the risk that it would shut off its own synthesis. In combination with the in vitro RNAP reconstitution technology (15,31,39), this system will hopefully allow us to obtain RNAP molecules defective in essential functions.…”

DNA-Dependent RNA Polymerases of the Archaebacteria

“…Dissociation and reconstitution approaches, which have helped in elucidating the role of the components of bacterial RNA polymerases (Heil and Zillig, 1970;Palm et al, 1975;Ishihama and Ito, 1972), have not yet been worked out for the eukaryotic RNA polymerases. In summary, eukaryotic and eubacterial RNA polymerases are clearly distinguished by component patterns and functional characteristics such as promoter recognition sites, which would be expected for homologous molecules from different primary kingdoms.…”