PMID: 6397224Dec 18, 1984Paper

Intrinsic zinc ion is essential for proper conformation of active Escherichia coli RNA polymerase

Biochemistry
D Solaiman, F Y Wu

Abstract

The DNA-dependent RNA polymerase (RPase) from Escherichia coli contained 2 mol of Zn/mol of holoenzyme (alpha 2 beta beta' sigma). An in vitro protocol involving sequential denaturation of RPase in 8 M urea and low pH (2.2), in the presence of 10 mM ethylenediaminetetraacetic acid (EDTA), was developed to completely remove the two intrinsic Zn ions. Subsequent reconstitution of the denatured, Zn-free RPase in the absence and presence of 10(-5) approximately 10(-4) M ZnCl2 yielded respectively the inactive apoenzyme and active (50 +/- 10%) RPase containing one Zn ion (rec-Zn1-RPase). Active rec-Cd1-RPase was similarly obtained when CdCl2 instead of ZnCl2 was used in the reconstitution. The use of 65Zn as a tracer in the two-step reconstitution procedure showed that the metal was incorporated into renatured enzyme only in the last step of reconstitution. The subunit location of the incorporated metal was identified to be in the beta subunit by the use of Affi-Gel Blue column chromatography of rec-Cd1-RPase. The analysis of apo- and rec-Zn1-RPases by sucrose density gradient sedimentation showed that the inactive apo-RPase appeared to be consisted of randomly folded protein species with S20,w values ranging from 5 to 18 S, while r...Continue Reading

References

Aug 1, 1977·Archives of Biochemistry and Biophysics·N GonzalezM J Chamberlin
Sep 9, 1977·Biochemical and Biophysical Research Communications·V D'AuroraD S Sigman
Feb 28, 1978·Biochemical and Biophysical Research Communications·V D'AuroraD S Sigman
Aug 28, 1979·Biochimica Et Biophysica Acta·J A MillerA P Kimball
Oct 23, 1973·Biochemistry·F Y Wu, C W Wu
Oct 1, 1971·Proceedings of the National Academy of Sciences of the United States of America·M C ScruttonD A Goldthwait
Jun 1, 1966·Proceedings of the National Academy of Sciences of the United States of America·J P Richardson
Apr 1, 1983·Proceedings of the National Academy of Sciences of the United States of America·J S HanasC W Wu

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Citations

Sep 1, 1990·Trends in Biochemical Sciences·N A Woychik, R A Young
Jun 8, 2002·Journal of Molecular Biology·Ronald S Johnson, Rebecca E Chester

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