Metal- and DNA-binding properties and mutational analysis of the transcription activating factor, B, of coliphage 186: A prokaryotic C4 zinc- finger protein

Abstract

Coliphage 186 B is a 72-amino acid protein belonging to the Ogr family of analogous transcription factors present in P2-like phage, which contain a Cys-X2-Cys-X22-Cys-X4-Cys presumptive zinc-finger motif. The molecular characterization of these proteins has been hampered by their insolubility, a difficulty overcome in the present study by obtaining B as a soluble cadmium- containing derivative (CdB). Atomic absorption spectroscopy showed the presence of one atom of cadmium per molecule of purified CdB. The UV absorption spectrum revealed a shoulder at 250 nm. characteristic of CysS- Cd(II) ligand-to-metal charge-transfer transitions, and the difference absorption coefficient after acidification (Δε248, 24 mM-1 cm -1) indicated the presence of a Cd(Cys S)4 center. Gel mobility shift analysis of CdB with a 186 late promoter demonstrated specific DNA-binding (K(D),(app) 3-4/μM) and the protein was shown to activate transcription in vitro from a promoter-reporter plasmid construct. The B DNA-binding site was mapped by gel shift and DNAase I cleavage protection experiments to an area between -70 and -43 relative to the transcription start site, coincident with the consensus sequence, GTTGT-N8-TNANCCA, from -66 to -47 of the 186 and P2 late promoters. Inactive B point mutants were obtained in the putative DNA- binding loop of the N-terminal zinc-finger motif and in a central region thought to interact with the Escherichia coli RNA polymerase α-subunit. A truncated B mutant comprising the first 53 amino acids (B1-53) exhibited close to wild-type activity, showed a DNA-binding affinity similar to that of the full-length protein, and could be reconstituted with either Cd or Zn. Gel permeation analysis revealed that B1-53 was a majority dimeric species whereas wild-type B showed larger oligomers. 186 B therefore exhibits a potentially linear organization of functional regions comprising an N terminal C4 zinc-finger DNA-binding region, a dispensable C-terminal region involved in protein self-association, and a central region that interacts with RNA polymerase.