Stabilization of the primary sigma factor of Staphylococcus aureus by core RNA polymerase
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The primary sigma factor (σA) of Staphylococcus aureus, a potential drug target, was little investigated at the structural level. Using an N-terminal histidine-tagged σA (His-σA), here we have demonstrated that it exits as a monomer in solution, possesses multiple domains, harbors primarily α-helix and efficiently binds to a S. aureus promoter DNA in the presence of core RNA polymerase. While both N- and C-terminal ends of His-σA are flexible in nature, two Trp residues in its DNA binding region are buried. Upon increasing the incubation temperature from 25º to 40ºC, ~60% of the input His-σA was cleaved by thermolysin. Aggregation of His-σA was also initiated rapidly at 45oC. From the equilibrium unfolding experiment, the Gibbs free energy of stabilization of His-σA was estimated to be +0.70 kcal mol-1. The data together suggest that primary sigma factor of S. aureus is an unstable protein. Core RNA polymerase however stabilized σA appreciably.
Abstract
The primary sigma factor (σA) of Staphylococcus aureus, a potential drug target, was little investigated at the structural level. Using an N-terminal histidine-tagged σA (His-σA), here we have demonstrated that it exits as a monomer in solution, possesses multiple domains, harbors primarily α-helix and efficiently binds to a S. aureus promoter DNA in the presence of core RNA polymerase. While both N- and C-terminal ends of His-σA are flexible in nature, two Trp residues in its DNA binding region are buried. Upon increasing the incubation temperature from 25º to 40ºC, ~60% of the input His-σA was cleaved by thermolysin. Aggregation of His-σA was also initiated rapidly at 45oC. From the equilibrium unfolding experiment, the Gibbs free energy of stabilization of His-σA was estimated to be +0.70 kcal mol-1. The data together suggest that primary sigma factor of S. aureus is an unstable protein. Core RNA polymerase however stabilized σA appreciably.
참고문헌 (26)
- Paget, M. S., "The sigma70 family of sigma factors", Genome Biol., 4, pp.203~ (2003)
- Borukhov, S., "RNA polymerase holoenzyme: structure, function and biological implications", Curr. Opin. Microbiol., 6, pp.93~100 (2003)
- Wen, Y. D., "Structural and functional properties of a Bacillus subtilis temperature-sensitive sigma(A) factor", Proteins, 40, pp.613~622 (2000)
- Severinova, E., "Domain organization of the Escherichia coli RNA poly merase sigma 70 subunit", J. Mol. Biol., 263, pp.637~647 (1996)
- Chopra , I., "Bacterial RNA polymerase: a promising target for the discovery of new antimicrobial agents", Curr. Opin. Investig. Drugs, 8, pp.600~607 (2007)
- Sau, S., "Inactivation of indispensable bacterial proteins by early proteins of bacteriophages: implication in antibacterial drug discovery", Curr. Protein Pept. Sci., 9, pp.284~290 (2008)
- Novick, R. P, "Rood, J. I", pp.496~516 (2006), ASM Press
- Somerville, G. A., "At the Crossroads of Bacterial Metabolism and Virulence Factor Synthesis in Staphylococci", Microbiol. Mol. Biol. Rev., 73, pp.233~248 (2009)
- Draghi, D. C., "Current antimicrobial resistance profiles among methicillin resistant Staphylococcus aureus encountered in the outpatient setting", Diagn. Microbiol. Infect. Dis., 55, pp.129~133 (2006)
- Bergendahl, V., "Luminescence Resonance Energy Transfer-Based High- Throughput Screening Assay for Inhibitors of Essential Protein-Protein Interactions in Bacterial RNA Polymerase", Appl. Environ. Microbiol., 69, pp.1492~1498 (2003)
- Deora, R., "Purification and characterization of DNA dependent RNA polymerase from Staphylococcus aureus", Biochem. Biophys. Res. Commun., 208, pp.610~616 (1995)
- Deora, R., "Characterization of the primary sigma factor of Staphylococcus aureus", J. Biol. Chem., 271, pp.21828~21834 (1996)
- Chanda, P. K., "Detection of antistaphylococcal and toxic compounds by biological assay systems developed with a reporter Staphylococcus aureus strain harboring a heat inducible promoter - lacZ transcriptional fusion", J. Biochem. Mol. Biol., 40, pp.936~943 (2007)
- Tyson, P. A., "Accessibility of tryptophan residues in Na, K-ATPase", J. Biol. Chem., 262, pp.4644~4648 (1987)
- Bohm, G., "Quantitative analysis of protein far UVcircular dichroism spectra by neural networks", Protein Eng., 5, pp.191~195 (1992)
- Greene, R. F. Jr., "Urea and guanidine hydrochloride denaturation of ribonuclease, lysozyme, alpha-chymotrypsin, and beta-lactoglobulin", J. Biol. Chem., 249, pp.5388~5393 (1974)
- Ganguly, T., "Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity", Virol. J., 4, pp.64~ (2007)
- Vassylyeva, M. N., "Purification, crystallization and initial crystallographic analysis of RNA polymerase holoenzyme from Thermus thermophilus. Acta Crystallogr", D. Biol. Crystallogr., 58, pp.1497~1500 (2002)
- Chang, B. Y., "Conformational properties of Bacillus subtilis RNA polymerase sigma A factor during transcription initiation", Biochem. J., 294, pp.43~47 (1993)
- Nagai, H., "Regions of the Escherichia coli primary sigma factor sigma70 that are involved in interaction with RNA polymerase core enzyme", Genes Cells, 2, pp.725~734 (1997)
- Ganguly, T., "Physicochemical properties and distinct DNA binding capacity of the repressor of temperate Staphylococcus aureus phage phi11", FEBS J., 276, pp.1975~1985 (2009)
- Sambrook, J., "Molecular Cloning: A Laboratory Manual. 3rd ed" (2001), Cold Spring Harbor Laboratory Press
- Ausubel, F. M., "Current Protocols in Molecular Biology" (1998), John Wiley & Sons, Inc.
- Malabika Das, "Moderately thermostable phage Φ11 Cro repressor has novel DNA-binding capacity and physicochemical properties", BMB reports, 42(3), pp.160~165 (2009), 한국생화학분자생물학회
- Wessel, D., "A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids", Analytical Biochemistry, 138, pp.141~143 (1984)
- Das, M., "Overexpression, purification and characterization of repressor of temperate S. aureus phage φ11", J. Biochem. Mol. Biol., 40, pp.740~748 (2007)