Structural and functional differences between the two intrinsic zinc ions of Escherichia coli RNA polymerase

Abstract

DNA-dependent RNA polymerase (RPase) from Escherichia coli contains 2 mol of intrinsic Zn(II)/mol of core enzyme (alpha 2 beta beta'). In techniques analogous to those employed with the Zn(II) metalloenzyme aspartate transcarbamoylase [Hunt, J. B., Neece, S. H., Schachman, H. K., & Ginsberg, A. (1984) J. Biol. Chem. 259, 14793-14803], we show that titration of core or holoRPase with 10 or 16 equiv, respectively, of the sulfhydryl reagent p-(hydroxymercuri)benzenesulfonate (PMPS) results in the facile release of 1 mol of Zn(II) [B-site Zn(II)] in a reaction totally reversible with the addition of excess thiol provided no metal chelator is present. If ethylenediaminetetraacetic acid (EDTA) is present, reversal of the PMPS-enzyme complex results in formation of a Zn1 RPase [A-site Zn(II)]. This enzyme retains full transcriptional activity relative to Zn2 RPase on both calf thymus (nonspecific) and T7 (sigma-dependent, specific) DNA templates. If the core enzyme-PMPS complex is incubated with a large excess of another metal such as Cd(II) followed by thiol treatment, a hybrid ZnACdB RPase is formed. Direct treatment of the enzyme with excess Cd(II) also gives rise to a hybrid ZnACdB RPase. Transcription by these enzymes is also comparable to that of the starting Zn2 enzyme. Isolation of in vivo synthesized Co2 RPase and Cd2 RPase and treatment of either enzyme with PMPS/EDTA results in formation of a CoA and CdA enzyme, respectively. Co(II)A and Cd(II)A enzymes show 123 and 76%, respectively, of the elongation rates on T7 DNA observed for the Zn(II) enzyme. Visible absorption spectroscopy of the Co2 enzyme exhibits four d-d transition bands positioned at 760 (epsilon = 800), 710 (epsilon = 900), 602 (epsilon = 1500), and 484 (epsilon = 4000) nm. In addition, two charge-transfer bands are found at 350 (epsilon = 9600) and 370 (epsilon = 9500) nm. Only the Co(II) ion bound at site A is associated with this unique set of intense d-d transitions. The positions and intensities of both the visible and charge-transfer bands of Co(II)A RPase approximate those shown by Co(II)-substituted metalloenzyme sites where the ligands are four S rather than mixed S,N or S,O sites.