Purification and in vitro characterization of the Serratia marcescens NucC protein, a zinc-binding transcription factor homologous to P2 Ogr.
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 P(nucA) and to the sequence upstream of the P2 late promoter P(F) is accompanied by DNA bending. NucC protects about 25 nucleotides of the P(F) 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 P(F) in vitro.
McAlister
Victor
V
Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia, USA.
Zou
Chao
C
Winslow
Robert H
RH
Christie
Gail E
GE
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012706:Serratia marcescens / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D014157:Transcription Factors; D014764:Viral Proteins; C017800:zinc-binding protein; C074897:ogr protein, Enterobacteria phage P2; D012321:DNA-Directed RNA Polymerases