Characterization of the arginine repressor from Salmonella typhimurium and its interactions with the carAB operator
| Autor: | John E. Houghton, Ahmed T. Abdelal, Chung-Dar Lu |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
DNA
Bacterial Salmonella typhimurium Operator Regions Genetic Transcription Genetic Arginine Operon Molecular Sequence Data Repressor DNA footprinting Random hexamer Biology Ligases Bacterial Proteins Structural Biology Centrifugation Density Gradient Deoxyribonuclease I Amino Acid Sequence Binding site Molecular Biology Base Sequence Nucleic acid sequence Gene Expression Regulation Bacterial Molecular biology Repressor Proteins Biochemistry Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor |
| Zdroj: | Journal of Molecular Biology. 225:11-24 |
| ISSN: | 0022-2836 |
| DOI: | 10.1016/0022-2836(92)91022-h |
| Popis: | Primer extension experiments showed that the argR gene, encoding the arginine repressor in Salmonella typhimurium, is transcribed from a single promoter that is negatively regulated by arginine. A repressor overproducing strain was constructed and the repressor was purified to homogeneity. Gel filtration, sedimentation and cross-linking studies established that the native repressor is a hexamer of identical 17,000 Mr subunits. Gel retardation experiments indicate that the apparent dissociation constant for repressor/carAB operator is 6 x 10(-12) M. These experiments showed that arginine is essential for binding of the repressor to the DNA and that pyrimidine nucleotides have no significant effect on this binding. These results indicate that the effect of pyrimidines on expression of the arginine sensitive "downstream" carAB promoter is not directly mediated by the arginine repressor. These experiments also suggest that a single hexamer binds to the carAB operator, which carries two previously defined "ARG box" sequences that characterize operators for arg genes. Gel retardation experiments with DNA fragments carrying the individual ARG boxes showed that both boxes are required for effective binding of the hexameric repressor to the operator, indicating that the ARG boxes comprise a single binding site for the repressor. Analysis of the potential secondary structure of the arginine repressor does not reveal any of the recognizable structural motifs common to a number of DNA-binding proteins. A combination of DNase I, premethylation interference, depurination and hydroxyl radical footprinting techniques were employed to characterize the interactions of the repressor with the carAB operator, with the results suggesting that the repressor predominantly interacts with A.T residues in this region. Comparative DNA sequence analysis of the known arginine operators of enteric bacteria further indicates that the specificity of interaction may be based more on the precise distance between two defined A.T-rich regions rather than on the specific nucleotide sequence. |
| Databáze: | OpenAIRE |
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