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, 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) and bacterial genetics are used in the laboratory to solve this challenging problem.

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.
6. Isolation of phage derived inhibitors of the transcription machinery.

Research Highlights

Interaction with the nascent RNA is a pre-requisite for the recruitment of Rho to the transcription elongation complex in vitro (JMB, 2011).

In the conventional model of the Rho-dependent transcription termination, the terminator Rho binds to the rut site (Rho utilization) and translocates along the nascent RNA prior to making possible interactions with the elongating RNA polymerase (RNAP). Even though the interaction between Rho and isolated RNAs were studied in great detail, the same has never been shown with the nascent RNA emerging from the transcription elongation complex (EC). Direct demonstration and requirement of the Rho-nascent RNA binding becomes even more important because of the recently proposed alternative model where Rho loads onto the RNAP prior to the formation of the nascent RNA. Here we have measured the direct association of Rho in vitro with the free RNAP, RNAP-promoter binary complex and stalled ECs with varied length of RNA. We observed the association of Rho only with the ECs having the rut site containing long nascent RNA. This association was significantly reduced when either a Rho mutant, Y80C, defective for RNA-binding, or an anti-sense oligo to the rut site was used, or rut site was eliminated by RNAse digestion, or replacing with a random RNA sequence. Presence of EC-bound NusG, the binding partner of Rho, did not facilitate this association. RNAse foot-printing of the Rho-EC complex revealed a clear Rho-mediated protection of the rut sites on the nascent RNA. We concluded that the nascent RNA loading of Rho and its interaction with the rut site is mandatory and a pre-requisite for its recruitment to the EC under in vitro experimental conditions.

Interaction surface of the transcription terminator Rho required to form complex with the C-terminal domain of the antiterminator NusG (JMB 2011).

The Rho-dependent transcription termination in bacteria requires the interaction between the terminator, Rho and the antiterminator, NusG. The interaction surface of the Rho-NusG complex is unknown. Here we provide direct evidence that the .-sheet bundle of the C-terminal domain of NusG (NusG-CTD) has the binding determinants for the Rho which proves the hypothesis described earlier (Mooney et al., 2009; ref. 16). Disulfide bridges can be engineered from the NusG-CTD with the surface exposed amino acids 217 and 224 of Rho, belonging to its P-loop ATPase domain. Mutational analyses of this region of Rho revealed that a hydrophobic pocket, located behind these amino acids of Rho, is the docking site for the NusG-CTD. Proximity of this region of Rho to NusG-CTD in the Rho-NusG complex was also confirmed by the efficient FRET between the residues K224 of Rho and A168 of NusG-CTD. The identification of the Rho-NusG interaction surface will be useful not only to understand the role of NusG in the termination process but also may explain the molecular basis of involvement of the NusG-CTD for recruiting the Rho and the ribosome to the same transcription machinery.

A bacterial transcription terminator with inefficient molecular motor action but with a robust transcription termination function (JMB 2010).

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.

Projects in progress

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

1. Fast kinetics approaches to follow termination processes using stopped flow/quench flow methods.
2. Design of transcription antiterminator.

Extramural Funding

1. DBT grant (2011 - 2014).
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.
6. 2011: Elected fellow of NASI, Allahabad.

Reviewer of Journals/grants:

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