Isolated Operator Binding and Ligand Response Domains of the TyrR Protein of Haemophilus influenzae Associate to Reconstitute Functional Repressor
| Autor: | Ronald L. Somerville, Shimin Zhao |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
DNA
Bacterial Operator Regions Genetic Protein Conformation Repressor Ligands Cleavage (embryo) Biochemistry Cofactor Plasmid Escherichia coli Molecular Biology Gene biology Ligand Circular Dichroism Escherichia coli Proteins Cell Biology beta-Galactosidase Haemophilus influenzae Molecular biology Peptide Fragments Protein tertiary structure Molecular Weight Repressor Proteins Cytoplasm biology.protein Electrophoresis Polyacrylamide Gel Dimerization Transcription Factors |
| Zdroj: | Journal of Biological Chemistry. 274:1842-1847 |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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