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Escherichia coli RNA polymerase is a metalloenzyme containing 2 g-atoms of tightly bound zinc per mol of enzyme. We have prepared RNA polymerase from E. coli cells grown in a zinc-depleted medium supplemented with cobalt(II) chloride. The purified enzyme contains 1.8-~2.2 g-atoms of cobalt per mol of enzyme with concomitant reduction in the zinc content. The cobalt-substituted enzyme is enzymatically as active as Zn-RNA polymerase on a variety of templates under standard assay conditions. These two enzymes are almost identical by such physical criteria as subunit composition, monomer-dimer equilibrium, and pH and temperature stabilities. They differ in that Co-RNA polymerase exhibits a visible absorption spectrum with two major peaks at 584 and 703 nm. Addition of nucleoside triphosphates selectively perturbs the 584-nm peak, whereas addition of a template analogue, d(pT)t0, affects both peaks. These spectral changes suggest that the tightly bound metal ions may directly or indirectly participate in binding of substrate or template to the enzyme. Biochemically, both enzymes are also very similar with respect to pH-activity profile, extrinsic metal require--Al variety of nucleotidyl transferases including DNA and RNA polymerases from both prokaryotic and eukaryotic sources have been shown to be zinc metalloenzymes (Slater et

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Equilibrium and kinetic studies of the interaction of rifampicin with RNA polymerase of Escherichia coli were performed by exploiting the quenching of intrinsic fluorescence of the protein by the drug. Fluorimetric titrations show that rifampicin binds stoichiometrically to the core and holoenzyme with an apparent Kd of less than or equal to 3 x 10(-9) M. Neither the addition of template nor the formation of the initiation complex in the presence of dinucleotide and nucleoside triphosphate prevents the rifampicin-enzyme interaction. Although the equilibrium binding constant for the rifampicin-RNA polymerase complex is about the same for the core and holoenzyme and the holoenzyme-T7 DNA complex, stopped-flow studies indicate that the rates at which rifampicin interacts with these enzyme forms are different. In all three cases, the kinetic data can be interpreted in terms of a mechanism in which the rapid bimolecular binding of rifampicin to RNA polymerase is followed by a relatively slow isomerization of the drug enzyme complex: (See article). While the values of dissociation constant K1 = (k-1/k1), for the first binary complex (ER) are similar, the rate constant for the forward isomerization, k2, decrease in the order of core enzyme greater than holoenzyme greater than the holoenzyme-T7 DNA complex. The fact that this order is parallel to the relative rates of inactivation of the enzymes and the enzyme-DNA complex suggests that the inactivation may be due to the rifampicin-induced isomerization (conformational change) of the enzyme. This is supported by our observations that an enzyme complex which is in the process of elongating RNA chains can still bind rifampicin, although the enzyme activity is not inhibited by such binding. The values of overall binding constants calculated from the kinetic parameters, 1-2 x 10(-9) M, are in good agreement with the values of the apparent Kd obtained from fluorimetric titrations and Ki determined by enzymatic assays. In addition, the observations that the formation of an initiation complex leads to a significant but not complete rifampicin-resistant RNA synthesis and the recent finding that rifampicin only partly inhibits the formation of the first phosphodiester bond in an abortive initiation of RNA chains are consistent with our kinetic mechansim, i.e., the existence of two forms of the rifampicin-RNA polymerase complex, only one of which is able to initiate the RNA chains.

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