Abstract
The RNA polymerase holoenzyme consists of five subunits, four of which form a ‘core’ enzyme responsible for the catalysis of the polymerization of the nucleoside triphosphates. The fifth (sigma factor) is the ‘initiation factor’ involved in the recognition of correct starting points on the DNA template for transcription. The rate of the specific initiation in holoenzyme is generally higher than that of the non-specific initiation in the core enzyme. Although holoenzyme is stable in free solution sigma factor is lost once transcription has started. The interaction of sigma factor and core enzyme was studied in a series of experiments using the thiol-group reagent p-chloromercuribenzoate and an RNA-synthesizing system in vitro. Pure core enzyme was prepared essentially as described by Chamberlin & Berg (1962) with phosphocellulose and zonal-rotor steps replacing the hydroxyapatite step. Pure holoenzyme was prepared with a single-stranded-DNA affinity-column step (Schaller et al., 1972) included and sigma factor content was determined by a rifampicin column. p-Chlor~[~~~Hg]merc~ribenzoate w s used to show that sigma factor contains three thiol residues/mol. Sigma factor was prepared (Burgess & Travers, 1971) and enzyme activity assayed by following the incorporation of radioactivity from [8-14C]ATP into acidinsoluble material. In our assay core enzyme was able to transcribe calf thymus DNA without the presence of sigma factor, but with bacteriophage T7 DNA as template sigma factor was required and gave typically a 40-fold stimulation. Hence, two integrated mechanisms are in operation: first, the catalysis by core enzyme of the polymerization reaction that takes place in the absence of sigma factor; secondly, the stimulation mediated by sigma factor of the recognition and initiation events. By using p-chloromercuribenzoate, experiments were designed to differentiate between the two systems. Inhibition of the polymerase reaction by p-chloromercuribenzoate has been demonstrated by using core enzyme and calf thymus DNA template (King & Nicholson, 1972). In a similar way core enzyme was treated with 1-8mol ofp-chloromercuribenzoate/mol and the enzyme assayed on a bacteriophage T7 DNA template. As expected, a similar curve to the calf thymus DNA case was obtained when percentage inhibition was compared. The addition of excess of thiols in the form of 2-mercaptoethanol to the p-chloromercuribenzoate-treated enzyme gave complete recovery of activity at all amounts of p-chloromercuribenzoate treatment. Hence, inhibition of the transcribing activity of the core enzyme was freely reversible. In a second experiment addition of sigma factor to core enzyme pretreated with p-chloromercuribenzoate followed by assay on calf thymus or bacteriophage T7 DNA templates gave percentage-inhibition curves which were superimposable for both templates and similar to those obtained in the first experiment. It therefore may be concluded that the polymerization function of core enzyme is inhibited and not its mediation of sigma factor function, since if core enzyme thiol groups were involved in this mediation then a rapid inhibition of synthesis on bacteriophage T7 DNA template would be expected when compared with synthesis on calf thymus DNA template. Confirmation of these results came from experiments on the restoration of activity of p-chloromercuribenzoate-treated core enzyme. Core enzyme with different numbers
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