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Journal of Bacteriology, April 2001, p. 2527-2534, Vol. 183, No. 8
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.8.2527-2534.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Cytoplasmic RNA Polymerase in Escherichia coli

N. Shepherd,1,dagger P. Dennis,2,* and H. Bremer1

Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas 75083-0688,1 and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada2

Received 6 November 2000/Accepted 26 January 2001

To obtain an estimate for the concentration of free functional RNA polymerase in the bacterial cytoplasm, the content of RNA polymerase beta  and beta ' subunits in DNA-free minicells from the minicell-producing Escherichia coli strain chi 925 was determined. In bacteria grown in Luria-Bertani medium at 2.5 doublings/h, 1.0% of the total protein was RNA polymerase. The concentration of cytoplasmic RNA polymerase beta  and beta ' subunits in minicells produced by this strain corresponded to about 17% (or 2.5 µM) of the value found in whole cells. Literature data suggest that a similar portion of cytoplasmic RNA polymerase subunits is in RNA polymerase assembly intermediates and imply that free functional RNA polymerase can form a small percentage of the total functional enzyme in the cell. On infection with bacteriophage T7, 20% of the minicells produced progeny phage, whereas infection in 80% of the cells was abortive. RNA polymerase subunits in lysozyme-freeze-thaw lysates of minicells were associated with minicell envelopes and were without detectable activity in an in vitro transcription assay. Together, these results suggest that most functional RNA polymerase is associated with the DNA and that little if any segregates into DNA-free minicells.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, BC V6T 173, Canada. Phone: (604) 822-5975. Fax: (604) 822-5227. E-mail: patrick.p.dennis{at}ubc.ca.

dagger Present address: GlaxoWellcome, Research Triangle Park, NC 27709.

Journal of Bacteriology, April 2001, p. 2527-2534, Vol. 183, No. 8
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.8.2527-2534.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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