Preparation and characterization of various Escherichia coli RNA polymerases containing one or two intrinsic metal ions.
The Escherichia coli DNA-dependent RNA polymerase (RPase) holoenzyme (alpha 2 beta beta' sigma) possesses 2 mol equiv of Zn: beta and beta' subunits each contain one Zn ion. An in vitro metal-substitution method developed earlier (method I) was used to remove the two intrinsic Zn ions and then to reconstitute other metal ions into the beta subunit of RPase. One Cd or Hg ion was successfully reconstituted into half-active enzymes (rec-Cd1- or rec-Hg1-RPase), while Mn or Ni ion was not incorporated. A new, simplified in vitro metal-substitution method (method II), which omitted the low-pH treatment and subsequent urea dialysis in method I, was devised in this study. Consequently, Zn or Cd could be incorporated into both the beta and beta' subunits, resulting in rec-Zn2- or rec-Cd2-RPase, respectively. However, only one Hg was incorporated, probably due to steric hindrance by the large size of the Hg ion, while Mn, Ni, or Cr was not bound by the reconstituted enzyme, which instead incorporated only one Zn. Analysis of the metal content of various reconstituted RPases indicated that without low-pH treatment Zn bound to both the beta and beta' subunits when Zn concentrations were higher than 2 X 10(-6)M, but it bound only to the beta' subunit at lower concentrations. Moreover, low-pH treatment destroys the metal binding site in the beta' subunit. The metal sites on the beta and beta' subunits did not have significant affinity for the transition metals such as Mn, Ni, and Cr.(ABSTRACT TRUNCATED AT 250 WORDS)
D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008667:Metalloproteins / Q000302:isolation & purification*; D008670:Metals / Q000032:analysis*; D010957:Plasmids; D013054:Spectrophotometry, Atomic / Q000379:methods; D013438:Sulfhydryl Compounds / Q000032:analysis
D008667:Metalloproteins; D008670:Metals; D013438:Sulfhydryl Compounds; D012321:DNA-Directed RNA Polymerases