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.The NucC protein was originally identified as a positive regulator of extracellular nuclease and bacteriocin 28b production in Serratia marcescens (7,12). NucC is a basic 75-aminoacid protein encoded by what appears to be a cryptic prophage and is a member of the bacteriophage P2 Ogr family of transcriptional activators. These proteins, which are required for late gene expression in P2-and P4-related phages, represent a novel class of prokaryotic zinc finger transcription factors. All of the members of this family are functionally interchangeable, at least to some extent, and have significant amino acid sequence similarity. Four invariant Cys residues, arranged in a Cys-X 2 -Cys-X 22 -Cys-X 4 -Cys motif, are essential for function and involved in the coordination of zinc (8,15,19,21,26). Binding sites for these proteins have been localized upstream of P2 and P4 late promoters and the S. marcescens nuclease promoter (1,10,13,14,33,34); these sites include a conserved element of hyphenated dyad symmetry, TGT-N 12 -ACA. Genetic evidence indicates that members of this family of proteins activate transcription via a specific interaction with the C-terminal domain of the ␣ subunit of RNA polymerase (2,31,35).In vitro analysis of the mechanism of transcription activation by members of the P2 Ogr family has been hampered by the relative insolubility of these proteins. The binding sites in P2 and P4 late promoters were defined by using protein mixtures obtained by copurification with a MalE fusion protein (13,14). The related 186B protein was purified as a Cd 2ϩ derivative that retained partial activity (26). We have succeeded in purifying highly active native NucC protein and report here the analysis of the DNA binding specificity and in vitro activity of purified NucC. MATERIALS AND METHODSBacterial strains and plasmids. The strains and plasmids used in this study are listed in Table 1.Plasmid construction and DNA manipulations. Standard methods wer...
The Serratia marcescens extracellular nuclease gene,nucA, is positively regulated by the product of thenucC gene. In this study, the upstream region required for NucC-dependent nuclease expression was defined by using fusions to the gene encoding chloramphenicol acetyltransferase (cat). This sequence includes an element of hyphenated dyad symmetry identified previously as the binding site for the P2 Ogr family of activators. Footprint analysis confirmed that members of this family of activator proteins bind to this site, protecting a region between −76 and −59 relative to the start of transcription. The activator binding site in the nucA promoter lies one turn of the helix upstream from the corresponding sites in the P2 and P4 late promoters. The effects of deletions between the downstream end of the activator binding site and the putative −35 region are consistent with a strict helical phasing requirement for activation.
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