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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 mycobacteriophage genomics approach to identify novel mycobacteriophage proteins having Mycobactericidal properties (Microbiology in press).

The Mycobacteriophages specific to mycobacteria are the sources of varieties of effector proteins capable of eliciting bactericidal responses. We describe a genomics approach combining with bioinformatics to identify mycobacteriophage proteins that are toxic to mycobacteria upon expression. A genomic library made from the collections of phage genomes is screened for the clones capable of killing the M. smegmatis strain mc2155. We identified four unique clones; clones 45 and 12N (from the mycobacteriophage D29), clones 66 and 85 (from the mycobacteriophage Che12). The gene products from the clones 66 and 45 were identified as Gp49 of Che12 phage and Gp34 of D29 phage, respectively. The gene products of the other two clones, 85 and 12N, utilized novel ORFs coding for synthetic proteins. These four clones (clone 45, 66, 85 and 12N) upon expression caused growth defects in M. smegmatis and M. bovis. Clones having Gp49 and Gp34 also induced growth defects in E. coli indicating that they target conserved host-machineries. Their expressions induced various morphological changes indicating that they affected DNA replication and cell division steps. We predicted Gp34 to be a Xis protein required in phage DNA excision from the bacterial chromosome. Gp49 is predicted to have a HTH motif having DNA-bending/twisting properties. We suggest that this methodology is useful to identify new phage proteins having desired properties without laboriously characterizing the individual phages. It is universal and could be applied to other bacteria-phage systems. We speculate that the existence of virtually “unlimited” number of phages and their unique gene products could offer cheaper and less hazardous alternative to explore new antimicrobial molecules.


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.

A bacteriophage capsid protein is an inhibitor of a conserved transcription terminator of various bacterial pathogens (J. Bact, 2018).

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.

Projects in progress:

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

Extramural Funding:

  1. Tata Innovation Fellowship (2019-2022).
  2. DST Grant (2016-2019).
  3. DBT grant (2019-2022).
  4. 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.
  9. 2018: Elected fellow IASc, Bangalore.
  10. 2018: Fellow of Telengana Academy of Sciences.
  11. 2019: DBT TATA Innovation Fellowship.

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 : Wednesday, 1th May, 2019.

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