Molecular Interactions between Two Global Regulators, sar and agr, in Staphylococcus aureus
| Autor: | Yueh-tyng Chien, Ambrose L. Cheung |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Staphylococcus aureus
Transcription Genetic RNAIII Recombinant Fusion Proteins DNA Mutational Analysis Molecular Sequence Data Biology Biochemistry DNA-binding protein Bacterial Proteins Transcription (biology) Direct repeat RNA Antisense Binding site Molecular Biology Gene Transcription factor Genetics Binding Sites Base Sequence Virulence Promoter Gene Expression Regulation Bacterial Cell Biology Molecular biology DNA-Binding Proteins RNA Bacterial Trans-Activators RNA Protein Binding Transcription Factors |
| Zdroj: | Journal of Biological Chemistry. 273:2645-2652 |
| ISSN: | 0021-9258 |
| Popis: | The expression of many virulence determinants in Staphylococcus aureus is controlled by regulatory loci such as agr and sar. We have previously shown that the SarA protein is required for optimal transcription of RNAII and RNAIII in the agr locus. To define the specific molecular interaction, we overexpressed SarA as a glutathione S-transferase (GST) fusion protein by cloning the 372-base pair (bp) sarA gene into the vector. The purified GST-SarA as well as cleaved SarA were able to bind specifically to the P2, P3, and the combined P2-P3 promoter fragments of agr in gel shift assays. Using monoclonal antibodies to SarA, we found that SarA is a part of the retarded protein-DNA complex as evidenced by the formation of a supershifted band. The SarA binding site on the agr promoter, mapped by DNase I footprinting assay, covered a 29-bp region between the P2 and P3 promoters devoid of any direct repeats. A synthetic 45-bp fragment encompassing the 29-bp sequence also bound the SarA protein in band shift assays. Serial in-frame deletion analysis of sarA revealed that, with the exception of 15 residues in the N terminus, almost all of SarA (residues 16-124) is essential for agr binding activity. Northern analysis confirmed that only the sar mutant clone containing a truncated sarA gene with a 15-residue deletion in the N terminus (SarA16-124) could activate agr transcription to a level approaching that of the full-length counterpart (SarA1-124). Taken together, these data indicated that SarA is a DNA-binding protein with binding specificity to the P2 and P3 interpromoter region of agr, thereby activating RNAII and RNAIII transcription. |
| Databáze: | OpenAIRE |
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