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Metal‐ and DNA‐binding properties and mutational analysis of the transcription activating factor, B, of coliphage 186: A prokaryotic C4 zinc‐finger protein
Author(s) -
Pountney Dean L.,
Tiwari Ravi P.,
Egan J. Barry
Publication year - 1997
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560060416
Subject(s) - zinc finger , promoter , microbiology and biotechnology , transcription (linguistics) , dna , rna polymerase , binding site , chemistry , sp1 transcription factor , electrophoretic mobility shift assay , zinc , mutant , biology , transcription factor , rna , biochemistry , gene , gene expression , linguistics , philosophy , organic chemistry
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‐X 2 ‐Cys‐X 22 ‐Cys‐X 4 ‐Cys presumptive zinc‐finger motif. The molecular character‐ ization 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 (Δϵ 248 , 24 mM −1 cm −1 ) indicated the presence of a Cd(Cys‐S), 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‐N 8 ‐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 motifandin 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 exhibitsa 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.