Ongoing projects

Regulation of transcription in Mycobacterium tuberculosis

 

In the case of M. tuberculosis, we aim to understand the molecular mechanisms that govern the transition between replicating and non-replicating states. Our current research emphasis is to understand the transcription initiation mechanism. Multiple regulatory mechanisms govern the activity of transcription initiation factors, also called sigma factors. The intracellular concentration of free, activated sigma factors dictate the cellular response to different environmental signals. This is mediated by transcriptional, translational and post-translational processes. Structural and functional studies on M. tuberculosis sigma factors provide an insight into the diverse regulatory mechanisms that govern sigma factor activity. These include auto-regulation (SigA, SigC), redox stimuli (SigL, SigK), activation by a proteolytic cascade (SigD) or fused regulatory domains (SigJ).

 

Text Box: Schematic of the mechanism by which RslA regulates σL.

References-

 

The fused Snoal_2 domain of the Mycobacterium tuberculosis sigma factor σJ modulates promoter recognition.

Goutam, K., Gupta, A. K. & Gopal, B.

Nucleic Acids Res 45:9760-9772. 2017

 

Structural basis for the redox sensitivity of the Mycobacterium tuberculosis SigK-RskA σ-anti-σ complex.

Shukla, J.K., Gupta, R., Thakur, K.G., Gokhale, R. S., Gopal, B.

Acta Cryst. D70: 1026-1036. 2014

Mycobacterium tuberculosis RsdA provides a conformational rationale for selective regulation of σ factor activity by proteolysis.

Jaiswal R K, Suryaprabha T, Manjeera G, Gopal B.

Nucleic Acids Res 41:3414-3423. 2013

Structural and biochemical basis for the redox sensitivity of Mycobacterium tuberculosis RslA

Thakur K G, Praveena T, Gopal B.

J. Mol. Biol.397, 1199-1208. 2010

Structural and biophysical studies on two promoter recognition domains of the extra-cytoplasmic function s factor sC from Mycobacterium tuberculosis.

Thakur K G, Joshi A M, Gopal B.

J. Biol. Chem 282, 4711-4718. 2008

 

The phenotypic switch from persistent to virulence in Staphylococcus aureus

 

Our emphasis in this area is on proteins involved in the quorum sensing mechanism. On-going projects in this area aim to understand the mechanim(s) by which RNA mediated regulatory mechanisms modulate the switch from the persistent (biofilm forming) to the virulent phenotype.

 

Text Box:  The S. aureus agr operon. A. Schematic of agr operon. B. Sequence features of P1, P2 and P3 promoter segments. C. The structure of AgrADBD domain in complex with the P3 promoter sites. AgrADBD interacts with promoter DNA by inserting loops into two successive major grooves and an intervening minor groove.    D. AgrADBD-P3_S1 promoter interactions.  The P3_S1 site is seen to be less engaged in interacting with AgrADBD with only one base specific interaction and few hydrogen bond interactions with the phosphate backbone. E. AgrADBD-P3_S2 promoter interactions. AgrADBD is involved in three base specific interactions with P3_S2

References-

A point mutation in AgrC determines cytotoxic or colonizing properties associated with phenotypic variants of ST22 MRSA strains.

Shambat, S.M., Siemens, N., Monk, I. R., Disha Mohan B, Mukundan, S. Krishnan, K. C., Prabhakara, S., Snall, J., Kearns, A., Vandenesch, F., Svensson, M., Kotb, M., Gopal, B., Arakere, G., Norrby-Teglund, A.

Sci Reports 6: 31360-31372. 2016

Influence of the AgrC-AgrA complex in the response time of Staphylococcus aureus quorum sensing.

Srivastava, S. K., Rajasree, K., Fasim, A., Arakere, G., Gopal, B.

J. Bacteriology 196: 2876-2888. 2014

The crystal structure of Staphylococcus aureus metallopeptidase (Sapep) reveals large domain motions between the manganese bound and apo states.

Girish T S , Gopal B.

J. Biol. Chem 285, 29406-29415. 2010

Molecular Basis for the role of Staphylococcus aureus Penicillin Binding Protein 4 in antimicrobial resistance.

Navratna V, Nadig S, Sood V, Prasad K, Arakere G, Gopal B.

J. Bacteriology 192: 134-144. 2010

 

Synthesis of peptide antibiotics

 

The current emphasis is on the synthesis of bacilysin. Bacilysin is a non-ribosomally synthesized dipeptide antibiotic that is active against a wide range of bacteria and some fungi. Synthesis of bacilysin (L-alanine-[2,3-epoxycyclohexano-4]-L-alanine) is achieved by proteins in the bac operon, also referred to as the bacABCDE (ywfBCDEF) gene cluster in Bacillus subtilis. Extensive genetic analysis from several strains of B. subtilis suggests that the bacABC gene cluster encodes all the proteins that synthesize the epoxyhexanone ring of L-anticapsin. Recently, we could demonstrate that BacA is a decarboxylase that acts on prephenate. Further, based on the biochemical characterization and the crystal structure of BacB, we note that BacB is anoxidase that catalyzes the synthesis of 2-oxo-3-(4-oxocyclohexa-2,5-dienyl)propanoic acid, a precursor to L-anticapsin. 

Text Box: Experimental evidence for a bound phenylpyruvate at the C-terminal active site of BacB. a, DNPH assay with denatured BacB extract. (○), DNPH alone; (■), phenylalanine treated with DNPH (negative control); (▴), phenylpyruvic acid-sodium salt treated with DNPH (positive control); (●), BacB treated with DNPH. b, ESI-spectrum of BacB extract in positive as well as in negative ion modes shows the presence of phenylpyruvic acid.

References-

Structural insights into the role of Bacillus subtilis YwfH (BacG) in tetrahydrotyrosine synthesis.

Rajavel M, Perinbam K, Gopal B.

Acta Cryst D69:324-332. 2013

Role of Bacillus subtilis BacB in the synthesis of the antibiotic bacilysin.

Rajavel M, Mitra A, Gopal B.

J. Biol. Chem 284:31882-31892. 2009

 

 

Other projects involve computational methods (Molecular Dynamics, modeling) or bioinformatics