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 C~S -X~-C~S -X~~-C~S -X , -C~S 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(I1) ligand-to-metal charge-transfer transitions, and the difference absorption coefficient after acidification 24 mM" cm") indicated the presence of a Cd(Cys-S), center. Gel mobility shift analysis of CdB with a 186 late promoter demonstrated specific DNA-binding (KD,app 3-4 pM) 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, G'ITGT-N,-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 a-subunit. A truncated B mutant comprising the first 53 amino acids (Bl-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 B 1-53 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.

Effect of static magnetic field onE. coli cells and individual rotations of ion-protein complexes

“…Transcription factors with zinc-®nger motif were well-characterized in different types of cells including prokaryotes [Pountney et al, 1997]. Therefore, there are several examples of the macromolecular rotations in E. coli cells covering the range of 4±300 rps.…”

Analysis of DNA Binding by a Eubacterial Zinc Finger Transcription Factor

“…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).…”

“…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.…”

A Carboxyl-terminal Cys2/His2-type Zinc-finger Motif in DNA Primase Influences DNA Content inSynechococcus PCC 7942

“…4B), but the latter remained substantially less than 1:1. It was initially hypothesized that this could be due to relative insolubility of the metallated-, compared with apo-, p10.5 since insolubility of a Zn 2ϩ -saturated prokaryotic Zn 2ϩ -binding protein expressed in E. coli has been reported (36). In previous studies of a metal-saturated prokaryotic protein containing a Cys 4 -type Zn 2ϩ -binding domain, such insolubility problems were overcome by using Cd 2ϩ as an analogue of Zn 2ϩ (36).…”

“…It was initially hypothesized that this could be due to relative insolubility of the metallated-, compared with apo-, p10.5 since insolubility of a Zn 2ϩ -saturated prokaryotic Zn 2ϩ -binding protein expressed in E. coli has been reported (36). In previous studies of a metal-saturated prokaryotic protein containing a Cys 4 -type Zn 2ϩ -binding domain, such insolubility problems were overcome by using Cd 2ϩ as an analogue of Zn 2ϩ (36). Cys 2 /His 2 zinc-fingers can bind one equivalent of Cd 2ϩ (37,38) and there are precedents for the metal ions being introduced either by in vitro exchange and/or following growth of E. coli cells in Cd 2ϩ supplemented medium (39).…”