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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. In vivo nature of Rho-dependent terminators.
  4. Physiological roles of Rho-dependent terminations.
  5. Super-resolution microscopy of the transcription machinery.
  6. Fast-kinetics approach to study the transcription termination processes.
  7. Isolation of mycobacteriophage derived proteins with antimicrobial activies.
  8. Design of antimicrobial peptides from bacteriophage proteins.

Collaborators::

  1. Prof Markus Wahl, Freie Universität Berlin, Germany (Cryo-EM).
  2. Prof. Udaydittya Sen, SINP, Kolkata (Crystallography).
  3. Prof. Agnieszka Szalewska-Palas, Uniwersytet Gdanski, Poland.

Research Highlights

Design of novel peptide-inhibitors against the conserved bacterial transcription terminator, Rho (JBC, 2021).

The transcription terminator Rho regulates many physiological processes in bacteria, such as antibiotic sensitivity, DNA repair, RNA-remodeling, etc, and hence, is a potential antimicrobial target, which is unexplored. The bacteriophage P4 capsid protein, Psu, moonlights as a natural Rho antagonist. Here, we report the design of novel peptides based on the C-terminal region of Psu using phenotypic screening methods. The resultant 38-mer peptides, in addition to containing mutagenized Psu sequences, also contained plasmid sequences, fused to their C-termini. Expression of these peptides inhibited the growth of E. coli, and specifically inhibited Rho-dependent termination in vivo. Peptides 16 and 33 exhibited the best Rho-inhibitory properties in vivo. Direct high-affinity binding of these two peptides to Rho also inhibited the latter's RNA-dependent ATPase and transcription termination functions in vitro. These two peptides remained functional even if 8-10 amino acids were deleted from their C-termini. In-silico modeling and genetic and biochemical evidence revealed that these two peptides bind to the primary RNA binding site of the Rho hexamer near its subunit interfaces. Additionally, the gene expression profiles of these peptides and Psu overlapped significantly. These peptides also inhibited the growth of Mycobacteria, and inhibited the activities of Rho proteins from M. tuberculosis, Xanthomonas, V. cholerae, and S. enterica. Our results showed that these novel anti-Rho peptides mimic the Rho-inhibition function of the ~42 kDa dimeric bacteriophage P4 capsid protein, Psu. We conclude that these peptides and their C-terminal deletion derivatives could provide a basis on which to design novel anti-microbial peptides (AMP).


The Rho-dependent transcription termination is involved in broad-spectrum antibiotic susceptibility in Escherichia coli (Front. Microbiology, 2020).

One of the major ways of acquiring multidrug resistance in bacteria is via drug influx and efflux pathways. Here, we show that E. coli with compromised Rho-dependent transcription termination function has enhanced broad-spectrum antibiotic susceptibility, which arises from the inefficient TolC-efflux process and increased permeability of the membrane. The Rho mutants have altered morphology, distinct cell surface, and increased levels of lipopolysaccharide in their outer membrane, which might have rendered the TolC efflux pumps inefficient. These alterations are due to the upregulations of poly-N-acetyl-glucosamine and lipopolysaccharide synthesis operons because of inefficient Rho functions. The Rho mutants are capable of growing on various dipeptides and carbohydrate sources, unlike their WT counterpart. Dipeptides uptake arises from the upregulations of the di-peptide permease operon in these mutants. The metabolomics of the Rho mutants revealed the presence of a high level of novel metabolites. Accumulation of these metabolites in these Rho mutants might titrate out the TolC-efflux pumps, which could further reduce their efficiency. We conclude that the transcription termination factor, Rho, regulates the broad-spectrum antibiotic susceptibility of E. coli through multipartite pathways in a TolC-dependent manner. The involvement of Rho-dependent termination in multiple pathways and its association with antibiotic susceptibility should make Rho-inhibitors useful in the anti-bacterial treatment regimen.


Rho-dependent transcription termination in bacteria recycles RNA polymerases stalled at DNA lesions (Nature Communications, 2019).

In bacteria, transcription-coupled repair of DNA lesions initiates after the Mfd protein removes RNA polymerases (RNAPs) stalled at the lesions. The bacterial RNA helicase, Rho, is a transcription termination protein that dislodges the elongation complexes. Here, we show that Rho dislodges the stalled RNAPs at DNA lesions. Strains defective in both Rho and Mfd are susceptible to DNA-damaging agents and are inefficient in repairing or propagating UV damaged DNA. In vitro transcription assays show that Rho dissociates the stalled elongation complexes at the DNA lesions. We conclude that Rho-dependent termination recycles stalled RNAPs, which might facilitate DNA repair and other DNA-dependent processes essential for bacterial cell survival. We surmise that Rho might compete with, or augment, the Mfd function.

Projects in progress:

  1. Nature of the Rho-dependent terminators in vivo.
  2. Understanding the physiological consequences of Rho-dependent termination.
  3. Understanding the role of the omega subunit of RNAP in Rho-dependent termination.
  4. In vivo localization of Rho and NusG by super-resolution microscopy.
  5. Identification Rho-RNAP functional interaction domains.
  6. Isolation and characterization of anti-mycobacterial proteins from mycobacteriophages.
  7. Design of antiterminator peptides from Psu protein.
  8. Computational approaches to understanding the conformational changes of Rho and NusG during the termination process.

Extramural Funding:

  1. DST-Indo-Polish Grant (2021-2022)
  2. Indo-German ICMR grant (2020-2023).
  3. DST-SERB Grant (2020-2023).
  4. DBT-Tata Innovation Fellowship (2019-2022).
  5. DBT grant (2019-2022).

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.
  9. 2018: Elected fellow IASc, Bangalore.
  10. 2018: Fellow of Telangana Academy of Sciences.
  11. 2019: DBT TATA Innovation Fellowship.

Editorial Board Member:

  1. Journal of Applied Genetics (microbial Genetics section).

Reviewer of Journals/grants/Thesis:

  1. Nature Communications, TIBS, Journal of Bacteriology, Journal of Molecular Biology, Microbiology, PLOS one, Biochemical transactions, Communication Biology, msphere, MEEGID, Microbial Genomics, Scientific Reports, Indian Journal of Biophysics and Biochemistry, Journal of Bioscience, etc.
  2. The reviewer of grants for different granting agencies like DBT, DST, DRDO, Indo-French programs, Marsden Fund, New Zealand. etc.
  3. The reviewer of the thesis of the Ph.D. students from renowned institutions like SINP, Kolkata; Bose Institute, Kolkata; IISc., Bangalore; IMTech, Chandigarh, HCU, JNU, etc.

Patents:

  1. 'NOVEL SYNTHETIC PEPTIDES'; Indian Patent Application No. 201841048582 filed on December 20, 2019.
Contact Information
Email: rsen<at>cdfd.org.in
Phone: +91-40-27216103
Fax: +91-40-27216006
Last updated on : Thursday, 27th May, 2021.

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