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Dr. Ranjan Sen
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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 foot-printing 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.

Projects:

  1. Mechanism of transcription termination by transcription termination factor Rho.
  2. Mechanism of conversion of terminator into an antiterminator by N.
  3. Cross-talk between Rho-dependent termination and other biological processes.
  4. Fast-kinetics approach to study the transcription termination processes.
  5. Isolation of myco-bacteriophage derived inhibitors of the Mycobacterium sp.
  6. Design of terminator /antiterminator peptides.

Research Highlights

Molecular Basis of NusG-mediated Regulation of Rho-dependent Transcription Termination in Bacteria (JBC, 2016)

The bacterial transcription elongation factor NusG stimulates the Rho-dependent transcription termination through a direct interaction with Rho. The mechanistic basis of NusG dependence of the Rho function is not known. Here, we describe Rho* mutants I168V, R221C/A, and P235H that do not require NusG for their termination function. These Rho* mutants have acquired new properties, which otherwise would have been imparted by NusG. A detailed analyses revealed that they have more stable interactions at the secondary RNA binding sites of Rho, which reduced the lag in initiating its ATPase as well as the translocase activities. These more stable interactions arose from the significant spatial re-orientations of the P, Q, and R structural loops of the Rho central channel. We propose that NusG imparts similar conformational changes in the central channel of Rho, yielding faster isomerization of the open to the closed hexameric states of the latter during its RNA-loading step. This acceleration stabilizes the Rho-RNA interactions at many terminators having suboptimal rut sites, thus making Rho-NusG interactions so essential in vivo. Finally, identification of the NusG binding sites on the Rho hexamer led us to conclude that the former exerts its effect allosterically.


Transcription elongation factor NusA is a general antagonist of Rho-dependent termination in Escherichia coli (JBC, 2016).

NusA is an essential protein that binds to RNA polymerase (RNAP) and also to the nascent RNA, and influences transcription by inducing pausing and facilitating the process of transcription termination/antitermination. Its participation in Rho-dependent transcription termination has been perceived, but the molecular nature of this involvement is not known. We hypothesized that as both Rho and NusA are RNA-binding proteins, and have the potential to target the same RNA, the latter is likely to influence the global pattern of the Rho-dependent termination. Analyses of the nascent RNA-binding properties and consequent effects on the Rho-dependent termination functions of specific NusA-RNA binding domain mutants revealed an existence of Rho-NusA direct competition for the overlapping nut (NusA-binding site) and rut (Rho-binding site) sites on the RNA. This leads to delayed entry of Rho at the rut site that inhibits the latter’s RNA release process. High density tiling micro-array profiles of these NusA mutants revealed that a significant number of genes, together with transcripts from intergenic regions are up-regulated. Interestingly, majority of these genes were also up-regulated when the Rho function was compromised. These are strong evidences for the existence of NusA-binding sites in different operons which are also the targets of Rhodependent terminations. Our data strongly argue in favor of a direct competition between NusA and Rho for the access of specific sites on the nascent transcripts in different parts of the genome. We propose that this competition enables NusA to function as a global antagonist of the Rho function, which is unlike its role as a facilitator of hairpin dependent termination.


N protein from lambdoid phages transform NusA into an antiterminator by modulating NusA- RNA polymerase flap domain interactions (NAR, 2015).

Interaction of the lambdoid phage N protein with the bacterial transcription elongation factor NusA is the key component in the process of transcription antitermination. A convex surface of E.coli NusA-NTD, located opposite to its RNA polymerase-binding domain (the β-flap domain), directly interacts with N in the antitermination complex. We hypothesized that this N-NusA interaction induces allosteric effects on the NusA-RNAP interaction leading to transformation of NusA into a facilitator of the antitermination process. Here we showed that mutations in β-flap domain specifically defective for N antitermination exhibited altered NusA-nascent RNA interaction and have widened RNA exit channel indicating an intricate role of flap domain in the antitermination. Presence of N, reoriented the RNAP binding surface of NusA-NTD, which changed its interaction pattern with the flap domain. These changes caused significant spatial rearrangement of the βflap as well as the β' dock domains to form a more constricted RNA exit channel in the N-modified elongation complex (EC), which might play key role in converting NusA into a facilitator of the N antitermination. We propose that in addition to affecting the RNA exit channel and the active center of the EC, β-flap domain rearrangement is also a mechanistic component in the N antitermination process.


Projects in progress

  1. Characterization of NusG dependent terminators.
  2. Identifications of in vivo role of transcription termination factor Rho and its partner NusG using genomics approaches.
  3. In vivo localization of Rho and NusG by fluorescence microscopy.
  4. Identification Rho-RNAP interaction domain.
  5. Isolation and characterization of anti-mycobacterial proteins from mycobacteriophages.
  6. Constructions of antiterminator peptides from Psu protein.
  7. Computational approaches to understand the conformational changes of Rho and NusG during the termination process.

Extramural Funding

  1. DST Grant (2016-2019).
  2. DBT grant (2016-2019).
  3. Grant from DBT COE on "Microbial Physiology" (2014-2019).

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:

  1. TIBS, Journal of Molecular Biology, Journal of Bacteriology, Science Reports, Microbiology, PLOS one, Indian Journal of Biophysics and Biochemistry, Journal of Bioscience etc.
  2. Reviewer of grants for different granting agencies like DBT, DST etc.
  3. Member of DST-task force in Biophysics, biochemistry, Microbiology.
Contact Information
Email: rsen<at>cdfd.org.in
Phone: +91-40-24749428
Fax: +91-40-24749448
Last updated on : Friday, 28th October, 2016.

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