Probing activation of the prokaryotic arginine transcriptional regulator using chimeric proteins.
The major transcription factors controlling arginine metabolism in Escherichia coli and Bacillus subtilis, ArgR and AhrC, respectively, are homologous multimeric proteins that form l -arginine-dependent DNA-binding complexes capable of repressing transcription of the biosynthetic genes (both), activating transcription of catabolic genes (AhrC only) or facilitating plasmid dimer resolution (both). Multimerisation and l -arginine binding are associated with the C-terminal 70-80 residues; the N-terminal regions contain a winged helix-turn-helix DNA-binding domain. We have constructed chimeric genes in which the sequences for the N and C-terminal domains have been swapped. The resultant chimeric proteins and their corresponding native proteins have been analysed for their ability to multimerise and bind DNA operator sites in an L-arginine-dependent fashion. Gel filtration and equilibrium sedimentation analysis are consistent with the formation of hexamers by all four proteins in the presence of L-arginine and at high protein concentrations (>100 nM monomer). The hexamer sedimentation coefficients suggest that there is a reduction in molecular volume upon binding L-arginine, consistent with a conformational change accompanying an allosteric activation of DNA-binding. In the absence of L-arginine or at lower protein concentrations, the hexamers are clearly in rapid equilibrium with smaller subunits, whose dominant species appear to be based on trimers, as expected from the crystal structure of the ArgR C-terminal fragment, with the exception of the ArgR-C chimera, which apparently dissociates into dimers, suggesting that in the intact protein the DNA-binding domains may have a significant dimeric interaction. The hexamer-trimer Kdis in the micromolar range, suggesting that trimers are the principal species at in vivo concentrations.DNA binding by all four proteins has been probed by gel retardation and DNase I footprinting analysis using all three types of naturally occurring operators: biosynthetic sites encompassing two 18 bp ARG boxes separated by 2 bp; biosynthetic sites containing two such boxes and a third 18 bp ARG box at a distance of 100 bp downstream, i.e. within the structural gene; and finally a catabolic operator which contains a single ARG box site. The data show that all four proteins bind to the operators at the expected regions in an L-arginine-dependent fashion. From the apparent affinities of the chimeras for each target site, there is no obvious sequence-specificity associated with the N-terminal domains; rather the data can be interpreted in terms of differential allosteric activation, including DNA binding in the absence of L-arginine.Remarkably, the proteins show apparent "anti-competition" in the presence of excess, specific DNA fragments in gel retardation. This appears to be due to assembly of an activated form of the protein, probably hexamers, on the operator DNA. The data are discussed in terms of the current models for the mode of action of both native proteins.
Holtham
C A
CA
N.C.M.H. Unit, School of Biological Sciences, University of Nottingham, Sutton Bonington, Leicestershire, LE12 5RD, UK.
Jumel
K
K
Miller
C M
CM
Harding
S E
SE
Baumberg
S
S
Stockley
P G
PG
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D019677:Artificial Gene Fusion; D001412:Bacillus subtilis; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D018983:DNA Footprinting; D004269:DNA, Bacterial; D003850:Deoxyribonuclease I; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009875:Operator Regions, Genetic; D011387:Prokaryotic Cells; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014461:Ultracentrifugation
C120339:AhrC protein, Bacillus subtilis; C087018:ArgR protein, Bacteria; C490177:ArgR protein, E coli; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D001120:Arginine; D003850:Deoxyribonuclease I