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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 Rho-NusG interaction in vivo and in vitro.
  3. Physiological roles of Rho-dependent terminations.
  4. Super-resolution microscopy of the transcription/antitermination machineries.
  5. Fast-kinetics approach to study the transcription termination processes.
  6. Isolation of myco-bacteriophage derived inhibitors of the Mycobacterium sp.
  7. Design of terminator /antiterminator peptides.

Research Highlights

A bacteriophage capsid protein is an inhibitor of a conserved transcription terminator of various bacterial pathogens (J. Bact, 2017).

Rho is a hexameric molecular motor that functions as a conserved transcription terminator in majority of the bacterial species, which is a potential drug target. Psu is a bacteriophage P4 capsid protein that inhibits E.coli Rho by obstructing its ATPase and translocase activities. Here, we explored the anti-Rho activity of Psu for the Rho proteins from different pathogens. Sequence alignment and homology modelling of Rho proteins from pathogenic bacteria revealed the conserved nature of the Psu-interacting regions in all these proteins. We chose Rho proteins from various pathogens like, Mycobacterium smegmatis, Mycobacterium bovis, Mycobacterium tuberculosis, Xanthomonas campestris, Xanthomonas oryzae, Corynebacterium glutamicum, Vibrio cholerae, Salmonella enterica and Pseudomonas syringae. The purified recombinant Rho proteins of these organisms showed variable rates of ATP hydrolysis on the poly (rC) as substrate and were capable of releasing RNA from the E. coli transcription elongation complexes. Psu was capable of inhibiting these two functions of all these Rho proteins. In vivo pull down assays revealed direct binding of Psu with many of these Rho proteins. In vivo expression of psu induced killing of M. smegmatis, M. bovis, X.campestris, and S.enterica, indicating Psu-induced inhibition of Rho proteins of these strains under physiological conditions. We propose that the “universal” inhibitory function of the Psu protein against the Rho proteins from both the gram-negative and gram-positive bacteria could be useful for designing peptides having anti-microbial functions, and these peptides could be a part of synergistic antibiotic treatment of the pathogens through compromising the Rho functions.


Rho Protein: Roles and Mechanisms (Ann. Rev. Microbiology, 2017).

At the end of the multistep transcription process, the elongating RNA polymerase (RNAP) is dislodged from the DNA template either at specific DNA sequences, called the terminators, or by a nascent RNA-dependent helicase, Rho. In Escherichia coli, about half of the transcription events are terminated by the Rho protein. Rho utilizes its RNA-dependent ATPase activities to translocate along the mRNA and eventually dislodges the RNAP via an unknown mechanism. The transcription elongation factor NusG facilitates this termination process by directly interacting with Rho. In this review, we discuss current models describing the mechanism of action of this hexameric transcription terminator, its regulation by different cis and trans factors, and the effects of the termination process on physiological processes in bacterial cells, particularly E. coli and Salmonella enterica Typhimurium.


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.


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.
  7. 2015: Member DST- SERB, task force.
  8. 2018: Elected Fellow INSA, New Delhi.

Reviewer of Journals/grants:

  1. Nature Communications, TIBS, Journal of Molecular Biology, Molecular Microbiology, 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.
Contact Information
Email: rsen<at>cdfd.org.in
Phone: +91-40-27216103
Fax: +91-40-27216006
Last updated on : Monday, 11th June, 2018.

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