METAL-BINDING AND DNA-BINDING PROPERTIES AND MUTATIONAL ANALYSIS OF THE TRANSCRIPTION ACTIVATING FACTOR-B, OF COLIPHAGE-186 - A PROKARYOTICC4-ZINC-FINGER PROTEIN

Coliphage 186 B is a 72-amino acid protein belonging to the Ogr family of analogous transcription factors present in P2-like phage, which co ntain a Cys-X(2)-Cys-X(22)-Cys-X(4)-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 obt aining B as a soluble cadmium-containing derivative (CdB). Atomic abso rption spectroscopy showed the presence of one atom of cadmium per mol ecule of purified CdB. The UV absorption spectrum revealed a shoulder at 250 nm, characteristic of CysS-Cd(II) ligand-to-metal charge-transf er transitions, and the difference absorption coefficient after acidif ication (Delta epsilon(248), 24 mM(-1) cm(-1)) indicated the presence of a Cd(Cys-S)(4) center. Gel mobility shift analysis of CdB with a 18 6 late promoter demonstrated specific DNA-binding (K-D,K-app 3-4 mu M) and the protein was shown to activate transcription in vitro from a p romoter-reporter plasmid construct. The B DNA-binding site was mapped by gel shift and DNAase I cleavage protection experiments to an area b etween -70 and -43 relative to the transcription start site, coinciden t with the consensus sequence, GTTGT-N-8-TNANCCA, from -66 to -47 of t he 186 and P2 late promoters. Inactive B point mutants were obtained i n the putative DNA-binding loop of the N-terminal zinc-finger motif an d in a central region thought to interact with the Escherichia coli RN A polymerase alpha-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 r evealed 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-f inger DNA-binding region, a dispensable C-terminal region involved in protein self-association, and a central region that interacts with RNA polymerase.