title: Isolated operator binding and ligand response domains of the TyrR protein of Haemophilus influenzae associate to reconstitute functional repressor.
abstract: Highly purified preparations of the TyrR protein of Haemophilus influenzae Rd undergo specific and limited proteolytic cleavage during storage at 4 degreesC to generate two fragments of 28 and 8 kDa. Under nondenaturing conditions, the two fragments remain tightly associated. Nicked TyrR is identical to full-length TyrR in its operator binding characteristics. The 8-kDa fragment containing amino acid residues 258-318 was separated from the 28-kDa fragment (residues 1-257) by gel filtration chromatography in the presence of 4 M urea. Upon renaturation, this fragment bound to operator with an affinity similar to that of full-length TyrR but was unresponsive to ligands that normally modulate operator binding (gamma-S-ATP and L-tyrosine). It was not possible to renature the urea-treated 28-kDa fragment. Highly purified soluble preparations of truncated TyrR containing residues 1-257 were obtained after the overexpression of a shortened form of the tyrR gene via a specific plasmid construct. By several criteria, this species had native secondary and tertiary structure. The 28-kDa fragment was unable to bind to operator but could reconstitute nicked TyrR when added to the renatured 8-kDa fragment, as shown by physical properties and responsiveness to cofactors in operator binding. When either the 28- or 8-kDa species was expressed in vivo, there was no detectable operator binding, as evaluated using a lacZ reporter system driven by the repressible aroF promoter. When the two fragments were co-expressed in a common cytoplasm, an operator-binding species was formed, as demonstrated through partial restoration of repression capability.
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
authors.forename: S
authors.initials: S
authors.lastname: Zhao
authors.forename: R L
authors.initials: RL
authors.lastname: Somerville
mesh_terms: D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006193:Haemophilus influenzae / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008970:Molecular Weight; D009875:Operator Regions, Genetic; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D008024:Ligands; D010446:Peptide Fragments; D012097:Repressor Proteins; D014157:Transcription Factors; C069256:TyrR protein, E coli; D001616:beta-Galactosidase