Projects:

1. Mechanism of transcription termination by transcription termination factor Rho.
2. Mechanism of transcription antitermination by the antiterminator protein, N.
3. Mechanism of inhibition of Rho dependent termination by Psu.
4. Physiological importance of Rho-dependent termination.
5. Fast-kinetics approach to study the transcription termination processes.

Research Highlights

The molecular motors like helicases / translocases are capable of translocating along the single stranded nucleic acids and unwinding DNA or RNA duplex substrates using the energy derived from their ATPase activity. Bacterial transcription terminator, Rho, is a hexameric helicase and releases RNA from the transcription elongation complexes by an unknown mechanism. It has been proposed, but not directly demonstrated, that kinetic energy obtained from its molecular motor action (helicase / translocase activities) is instrumental in dissociating the transcription elongation complex. Here we report a hexameric Rho analogue (Rv1297, M. tb Rho) from Mycobacterium tuberculosis having poor RNA-dependent ATP hydrolysis and inefficient DNA:RNA unwinding activities. However, compared to the E.coli Rho it exhibited very robust and earlier transcription termination from the elongation complexes of the E.coli RNA polymerase. Bicyclomycin, an inhibitor of ATPase as well as RNA release activities of the E.coli Rho, inhibited the ATPase activity of the M. tb Rho with comparable efficiency but was not efficient in inhibiting its transcription termination function. Unlike E.coli Rho, the M. tb Rho was capable of releasing RNA in the presence of non-hydrolysable analogues of ATP quite efficiently. Also this termination function most likely does not require NusG, an RNA-release facilitator, as this Rho was incapable of binding to the NusG either of M. tb (Rv0639) or E.coli. These results strongly suggest that ATPase activity of the M. tb. Rho is uncoupled from its transcription termination function and this function may not be dependent on its helicase / translocase activity.

Rho-dependent transcription termination is an essential function in prokaryotes and the transcription terminator Rho is highly conserved among different species. Bacteriophage P4 capsid-decoration protein, Psu, specifically interacts with and inhibits the function of E.coli Rho. Interaction surface of Psu involved in interacting with Rho is not known, knowledge of which is not only important to understand the mechanism of its action but also will be useful to design peptide inhibitor(s) for Rho. We have isolated and characterized seven Psu mutants defective in interacting with Rho and in exerting anti-Rho activity. Conformational probing of Psu revealed that the N-terminal region of the protein folds over onto its central part forming a globular domain leaving a solvent exposed tail in the C-terminus. The mutations are located in both of these two domains. N-terminal mutants are instrumental in disrupting the N- to C-terminal cross talk in Psu which is required for its structural integrity and its function. Site-specific cross-linking experiments showed that the C-terminal tail preferentially cross-links to Rho and also this region of Psu was protected from limited proteolysis when bound to Rho. Therefore, the mutations in this region may have affected the direct interaction of Psu with Rho. We propose that the globular N-terminal domain of Psu confers structural integrity to the functionally important C-terminal tail which directly interacts with the hexameric Rho.

RNA polymerase (RNAP) pauses at different DNA sequences during transcription elongation and this pausing is associated with distinct conformational state(s) of the elongation complex (EC). Transcription termination e termination factor Rho, an RNA-dependent molecular motor, requires pausing of the EC in the termination zone of Rho-dependent terminators. We hypothesized that the conformational state(s) of the EC associated with this pausing would influence the action of Rho. Analyses of the pausing behavior of the EC at the termination points of two well-known Rho-dependent terminators revealed that Rho prefers actively transcribing complexes for termination. RNA release kinetics from stalled ECs showed that the rate of RNA release by Rho was reduced if the EC was irreversibly backtracked, if its RNA exit channel was modified by an RNA hairpin or the bridge helix/trigger loop movement in its active site was perturbed. These defects were overcome significantly by enhancing the rate of ATP hydrolysis either by increasing the concentration of ATP or by using a Rho mutant with higher ATPase activity. We propose that the force generated from ATP hydrolysis of Rho is the key factor in dislodging the EC through its molecular motor action, and this process is facilitated when the EC is in a catalytically competent state, undergoing rapid "Brownian ratchet" motion.

Projects in progress

1. Rho-NusG interaction.
2. Rho-Psu interaction.
3. Mechanism of Antitermination of Rho-dependent termination by N protein.
4. Characterization of Rho and NusG from M.Tb.
5. In vivo role of transcription termination factor Rho using genomics approaches.
6. Roles of NusA in transcription termination and antitermination.

1. Fast kinetics approaches to follow termination processes using stopped flow/quench flow methods.

Extramural Funding

1. DBT grant (2007-2010).
2. Grant from DBT COE on "Microbial Physiology" (2008-2013).
3. DST Swarnajayanti Fellowship (2008-2013)

Awards/Recognition:

1. 2002-2007: GRIP research grant award from NIH, USA.
2. 2003-2008: Wellcome Trust, UK, Senior Research Fellowship.
3. 2007: DBT Bioscience carrier development award.
4. 2007: Elected member of GRC.
5. 2008: DST Swarnajayanti Research Fellowship.

Reviewer of Journals/grants:

Journal of Molecular Biology, Indian Journal of Biophysics and Biochemistry etc.
Reviewer of grants for different granting agencies like DBT, DST etc.