Selective substitution in vitro of an intrinsic zinc of Escherichia coli RNA polymerase with various divalent metals.
A simple in vitro substitution method involving a sequential denaturation--reconstitution process was developed to substitute selectively one of the two intrinsic Zn ions in Escherichia coli RNA polymerase with Co, Mn, Ni, or Cu ion. The resultant metal hybrid Co-Zn, Mn-Zn, Ni-Zn, and Cu-Zn RNA polymerases possess 100, 100, 60, and 17% of the enzymatic activity of the reconstituted Zn-Zn enzyme, respectively. The substituted metal was found to be located in the beta subunit of the polymerase which contains the substrate binding site. The biochemical and physical properties of these metal-substituted polymerases were compared with those of the native Zn enzyme. Co-Zn and Ni-Zn core polymerases exhibit characteristic absorption spectra in the near-UV and visible region, while Mn-Zn and Cu-Zn enzymes do not. The Co-Zn enzyme shows two major peaks at 400 nm (epsilon = 3000) and 475 nm (epsilon = 2700), while the Ni-Zn enzyme exhibits a major peak at 462 nm (epsilon = 8000). The difference absorption spectrum of Ni-Zn core polymerase could be perturbed by the addition of substrate ATP but not by UTP in the absence of template and Mg(II) ion. These observations suggest that the substituted metal was located at the initiation site of the enzyme. The various metal hybrid enzymes do not differ appreciably in their abilities to incorporate noncomplementary nucleotide or deoxyribonucleotide into RNA product. It was found, however, that the difference in enzymatic activities of these metal hybrid enzymes resides at least partly in the initiation step of RNA synthesis.
D002413:Cations, Divalent; D003035:Cobalt / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology*; D009532:Nickel / Q000494:pharmacology; D011485:Protein Binding; D013053:Spectrophotometry; D015032:Zinc / Q000378:metabolism* / Q000494:pharmacology
D002413:Cations, Divalent; D008670:Metals; D003035:Cobalt; D008345:Manganese; D003300:Copper; D009532:Nickel; D012321:DNA-Directed RNA Polymerases; D015032:Zinc