Analysis of DNA Binding by a Eubacterial Zinc Finger Transcription Factor

Abstract

The Serratia marcescens NucC protein is structurally and functionally homologous to the P2 Ogr family of eubacterial zinc finger transcription factors required for late gene expression in P2-and P4-related bacteriophages. These activators exhibit site-specific binding to a conserved DNA sequence, TGT-N 3 -R-N 4 -Y-N 3 -aCA, that is located upstream of NucC-dependent S. marcescens promoters and the late promoters of P2-related phages. In this report we describe the interactions of NucC with the P2 FETUD late operon promoter P F . NucC is shown to bind P F as a tetramer and to make 12 symmetrical contacts to the DNA phosphodiester backbone. The Serratia marcescens NucC protein was originally identified as a positive regulator of extracellular nuclease and bacteriocin 28b production (15, 23). NucC is a basic protein of 75 amino acids that is a member of the bacteriophage P2 Ogr family, a novel class of eubacterial zinc-binding transcription factors required for late gene expression in P2-and P4-related phages. All members of this family characterized thus far are at least somewhat functionally interchangeable and have significant amino acid sequence similarity in the N-terminal twothirds of the protein. This similarity includes four essential Cys residues that are involved in coordination of zinc (17,27,30,36), arranged in a Cys-X 2 -Cys-X 22 -Cys-X 4 -Cys motif. The function(s) of the nonconserved C-terminal residues is unclear. Viable C-terminal deletions of P2 ogr and phage 186 B suggest that this region is dispensable, although it likely contributes to full activity (18,36). Genetic evidence is consistent with a mechanism for transcription activation that involves a direct interaction with the C-terminal domain of the ␣ subunit(s) of RNA polymerase (1,45,50). Purified NucC, RNA polymerase holoenzyme, and template DNA have been shown to be the only necessary components for open complex formation and transcription from the P2 late promoter P F, and NucC induces a 90°bend in DNA containing the P F binding site (35).Zinc fingers constitute a well-characterized motif for protein-DNA interaction, and structural data are available for a number of eukaryotic zinc finger transcription factors (reviewed in references 28, 29, and 32). Zinc finger proteins containing C 2 H 2 zinc coordination motifs represent the largest group of transcription factors in eukaryotes (38, 47). These proteins typically contain several zinc fingers that wrap around the DNA in a spiral manner. The zinc coordinating motif in each finger contains an ␣-helix preceded by a ␤-hairpin. Amino acid residues on the surface of the ␣-helix make contacts to adjacent bases in the major groove, and in most cases the ␤-hairpin makes phosphate backbone contacts. Two other types of zinc coordination motifs are exemplified by the yeast Gal4 protein and the hormone receptor family of transcriptional activators. Gal4 binds as a homodimer to the sequence CCG-N 11 -CGG (4, 7, 19). The zinc coordinating motif is composed of a pair of ␣-helices that coordinate two zin...

Deoxyuracil in DNA and disease: Genomic signal or managed situation?

“…Substitution of thymine for uracil in transcription factor binding sites has been shown to modify transcription factor binding in vitro (79,80) (Table 3). Substitution of a T:A pair with a U:A pair in the cAMP response element (CRE) decreases binding of cAMP response element binding protein (CBP) in vitro, while substitution of C:G with a U:G pair increases binding affinity for CBP (80).…”

“…It is still unclear whether or not uracil in DNA, or its excision, induce meaningful changes in gene expression. In vitro transcription factor binding studies have revealed changes in binding capacity following uracil substitution (79,80), but in vivo studies are required.…”