|Current Research Interests
Laboratory of transcription is engaged in understanding the mechanism of transcription termination and antitermination in prokaryotes. A wide range of techniques from biophysics (spectroscopy, thermodynamics, fast kinetics etc.), biochemistry (protein purification, chemical and enzymatic footprinting of protein and nucleic acids, cross-linking etc.), molecular biology (recombinant DNA techniques, site-directed mutagenesis), bacterial genetics and genomics are used in the laboratory to solve these intellectually challenging problems.
- Mechanism of transcription termination by transcription termination factor Rho.
- Mechanism of transcription antitermination by the antiterminator protein, N.
- Physiological importance of Rho-dependent termination.
- Fast-kinetics approach to study the transcription termination processes.
- Isolation of phage derived inhibitors of the transcription machinery.
- Design of terminator /antiterminator peptides.
The interaction surface of a bacterial transcription elongation factor required for complex formation with an antiterminator during transcription antitermination (JBC, 2013).
The bacterial transcription elongation factor, NusA, functions as an antiterminator when it is bound to the lambdoid phage derived antiterminator protein, N. The mode of N-NusA interaction is unknown, knowledge of which is essential to understand the antitermination process. It was reported earlier that in the absence of the transcription elongation complex (EC), N interacts with the C-terminal AR1 domain of NusA. However, the functional significance of this interaction is obscure. Here we identified mutations in NusA-N-terminus (NTD), specifically defective for N-mediated antitermination. These are located at a convex surface of the NusA-NTD, situated opposite to its concave RNA polymerase (RNAP) binding surface. These NusA mutants disrupt the N-nut site interactions on the nascent RNA emerging out of a stalled EC. In the N/NusA-modified EC, a Cys-53(S53C) from the convex surface of the NusA-NTD forms a specific disulfide (S-S) bridge with a Cys-39 (S39C ) of the NusA-binding region of the N protein. We conclude that when bound to the EC, the N- interaction surface of NusA shifts from the AR1 domain to its NTD domain. This occurred due to a massive away-movement of the adjacent AR2 domain of NusA upon binding to the EC. We propose that the close proximity of this altered N-interaction site of NusA to its RNAP-binding surface, enables N to influence the NusA-RNAP interaction during transcription antitermination that in turn facilitates the conversion of NusA into an antiterminator.
Structural and mechanistic basis of antitermination of Rho-dependent transcription termination by bacteriophage P4 capsid protein Psu (NAR, 2013).
The conserved bacterial transcription terminator, Rho, is a potent target for bactericidal agents. Psu, a bacteriophage P4 capsid protein, is capable of inducing antitermination to the Rho-dependent transcription termination. Knowledge of structural and mechanistic basis of this antitermination is required to design peptide-inhibitor(s) of Rho from Psu. Using suppressor genetics, cross-linking, protein foot-printing, and FRET analyses, we describe a conserved disordered structure, encompassing 139-153 amino acids of Rho, as the primary docking site for Psu. Also a neighbouring helical structure, comprised of 347-354 amino acids, lining its central channel, plays a supportive role in the Rho-Psu complex formation. Based on the crystal structure of Psu, its conformation in the capsid of the P4 phage, and its interacting regions on Rho, we build an energy-minimised structural model of the Rho:Psu complex. In this model, a V-shaped dimer of Psu interacts with the two diagonally opposite subunits of a hexameric Rho, enabling Psu to form a "lid" on the central channel of the latter. We show that this configuration of Psu makes the central channel of Rho inaccessible, and causes a mechanical impediment to its translocase activity.
The First Structure of Polarity Suppression Protein, Psu from Enterobacteria Phage P4, Reveals a Novel Fold and a Knotted Dimer (JBC, 2012).
Psu is a capsid decoration protein of bacteriophage P4 and acts as an antiterminator of Rho-dependent transcription termination in bacteria. So far, no structures have been reported for the Psu protein or its homologues. Here, we report the first structure of Psu solved by the Hg2_ single wavelength anomalous dispersion method, which reveals that Psu exists as a knotted homodimer and is first of its kind in nature. Each monomer of Psu attains a novel fold around a tight coiled-coil motif. CD spectroscopy and the structure of an engineered disulfidebridged Psu derivative reveal that the protein folds reversibly and reassembles by itself into the knotted dimeric conformation without the requirement of any chaperone. This structure would help to explain the functional properties of the protein and can be used as a template to design a minimal peptide fragment that can be used as a drug against Rho-dependent transcription termination in bacteria.
A multipronged strategy of an anti-terminator protein to overcome Rho-dependent transcription termination (NAR 2012).
One of the important role of Rho-dependent transcription termination in bacteria is to prevent gene expressions from the bacteriophage DNA. The transcription anti-termination systems of the lambdoid phages have been designed to overcome this Rho action. The anti-terminator protein N has three interacting regions, which interact with the mRNA, with the NusA and with the RNA polymerase. Here, we show that N uses all these interaction modules to overcome the Rho action. N and Rho co-occupy their overlapping binding sites on the nascent RNA (the nutR/tR1 site), and this configuration slows down the rate of ATP hydrolysis and the rate of RNA release by Rho from the elongation complex. N-RNA polymerase interaction is not too important for this Rho inactivation process near/at the nutR site. This interaction becomes essential when the elongation complex moves away from the nutR site. From the unusual NusA-dependence property of a Rho mutant E134K, a suppressor of N, we deduced that the N-NusA complex in the anti- termination machinery reduces the efficiency of Rho by removing NusA from the termination pathway. We propose that NusA-remodelling is also one of the mechanisms used by N to overcome the termination signals.
Projects in progress
- Role of NusG in Rho-dependent termination.
- Mechanism of Antitermination of Rho-dependent termination by N protein.
- Characterization of NusG from M.Tb.
- In vivo role of transcription termination factor Rho and its partner NusG using genomics approaches .
- Role of NusA in termination and antitermination.
- Creation of genomic library from bacteriophages.
- Characterization of hypothetical Rho-binding proteins.
- Constructions of terminator/antiterminator peptides.
- DBT Grant, as Co-PI (2013-2016).
- DBT grant (2011-2014).
- Grant from DBT COE on "Microbial Physiology" (2008-2013; 2013-2018).
- DST Swarnajayanti Fellowship (2008-2013).
- 2002-2007: GRIP research grant award from NIH, USA.
- 2003-2008: Wellcome Trust, UK, Senior Research Fellowship.
- 2007: DBT Bioscience carrier development award.
- 2007: Elected member of GRC.
- 2008: DST Swarnajayanti Research Fellowship.
- 2011: Elected fellow of NASI, Allahabad.
Reviewer of Journals/grants:
Journal of Molecular Biology, PLOS one, Indian Journal of Biophysics and Biochemistry, Journal of Bioscience etc.
Reviewer of grants for different granting agencies like DBT, DST etc.