The Modification of DNA‐Dependent RNA Polymerase from Escherichia coli by an Alkylating Derivative of Rifamycin SV

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Abstract
3-(2-Bromo[1-14C]acetamidoethyl)-thio-rifamycin SV, abbreviated BrAcNEtS-Rif, an alkylating derivative of rifamycin SV was synthesized. A four-fold excess of BrAcNEtS-Rif inhibited the enzymic activity of RNA polymerase from Escherichia coli to 97%. Incubation of RNA polymerase with Br[14C]AcNEtS-Rif led to covalent substitution. The reaction of Br[14C]AcNEtS-Rif with enzyme at a ratio of 1.4:1 and a concentration of 63 nM was found to proceed with a half life of 1 h at 37°C. The enzyme could be protected from reaction with BrAcNEtS-Rif by either rifampicin or the hybrid [poly(dT)] · [r(Ap)5A]. The modification of holoenzyme by Br[14C]AcNEtS-Rif in the presence of p-hydroxymercuribenzene sulfonic acid (pOH-HgBzSO3H) or 4 M LiCl occurred with faster kinetics and led to a higher degree of substitution. Reaction of Br[14C]AcNEtS-Rif with RNA polymerase core enzyme caused predominant substitution of subunit β. In the case of RNA polymerase holoenzyme the radioactive substituents were evenly distributed between subunits β and σ. Apparently the topology of the rifamycin binding site of holoenzyme, similarly to core enzyme, precludes attacks of nucleophilic functions from β' and α, but it allows nucleophilic functions from subunits β and σ to react with equal probability on BrAcNEtS-Rif. In the presence of a 20-fold excess of pOH-HgBzSO3H, the modification of holoenzyme was drastically altered. Virtually all substitution took place on subunit β', very little on β and none on subunits σ and α.