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Journal of Bacteriology, March 2003, p. 1808-1816, Vol. 185, No. 6
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.6.1808-1816.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Purification and In Vitro Characterization of the Serratia marcescens NucC Protein, a Zinc-Binding Transcription Factor Homologous to P2 Ogr

Victor McAlister, Chao Zou,{dagger} Robert H. Winslow, and Gail E. Christie*

Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia

Received 28 March 2002/ Accepted 16 December 2002

NucC is structurally and functionally homologous to a family of prokaryotic zinc finger transcription factors required for late gene expression in P2- and P4-related bacteriophages. Characterization of these proteins in vitro has been hampered by their relative insolubility and tendency to aggregate. We report here the successful purification of soluble, active, wild-type NucC protein. Purified NucC exhibits site-specific binding to a conserved DNA sequence that is located upstream of NucC-dependent Serratia marcescens promoters and the late promoters of P2-related phages. This sequence is sufficient for binding of NucC in vitro. NucC binding to the S. marcescens nuclease promoter PnucA and to the sequence upstream of the P2 late promoter PF is accompanied by DNA bending. NucC protects about 25 nucleotides of the PF upstream region from DNase I digestion, and RNA polymerase protects the promoter region only in the presence of NucC. Template DNA, RNA polymerase holoenzyme, and purified NucC are the only macromolecular components required for transcription from PF in vitro.

* Corresponding author. Mailing address: P.O. Box 980678, Richmond, VA 23298-0678. Phone: (804) 828-9093. Fax: (804) 828-9946. E-mail: christie{at}hsc.vcu.edu.

{dagger} Present address: Department of RARD, Aventis Pharmaceuticals, Bridgewater, NJ 08807.

Journal of Bacteriology, March 2003, p. 1808-1816, Vol. 185, No. 6
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.6.1808-1816.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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