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Molecular machines manage the flow of genetic information in DNA to active and functional proteins through the process of central dogma. The machines that mediate the information transfer from DNA to protein are RNA polymerase and ribosome. The synthesis of RNA from a DNA template is catalysed by RNA polymerase and the process is known as transcription. On the other hand, the process of information transfer from RNA to protein is mediated by ribosome. Our laboratory, since several decades has been working on the mechanistic details of transcription process in bacteria under stress.

The Lab focusses on the following questions:

  • Under nutritional starvation bacteria elicit 'stringent response' with concomitant synthesis of alarmone like (p)ppGpp. We focus our attention on how ppGpp modulate differential gene expression to balance the efficient utilization of available energy within the cell.
  • The second messenger like c-di-GMP or c-di-AMP play major role in bacterial communication network known as Quorum sensing. The proximate aim of our lab is to monitor the cross talk between stringent response and Quorum sensing.
  • RNA polymerase is a multi-subunit enzyme and every subunit is essential except omega, which can be deleted without any deleterious effect. However, omega plays a major role in maturation of the enzyme and maintaining its structural integrity. We are trying to find out through critical genetic experiments, the structure-function relationship in omega.
  • Lastly, we devote our time to characterise yet another protein Dps, which is the iron store house in bacteria and is synthesized under stress like starvation.

Recent Publications

Substrate-induced domain movement in a bifunctional protein DcpA regulates cyclic‑di‑GMP turnover: Functional implications of a highly conserved motif

Journal of Biological Chemistry

In eubacteria, cyclic-di-GMP (c-di-GMP) signaling is involved in virulence, persistence, and motility and generally orchestrates multicellular behavior in bacterial biofilms. Intracellular c-di-GMP levels are maintained by the opposing activities of diguanylate cyclases (DGCs) and cognate phosphodiesterases (PDEs). C-di-GMP homeostasis in Mycobacterium smegmatis is supported by DcpA, a conserved, bifunctional protein with both DGC and PDE activities. DcpA is a multi-domain protein whose GAF-GGDEF-EAL domains are arranged in tandem and are required for these two activities. To gain insight into how interactions among these three domains affect DcpA activity, here we studied its domain dynamics using real-time FRET. We demonstrate that substrate binding in DcpA results in domain movement that prompts a switch from an “open” to a “closed” conformation and alters its catalytic activity. We found that a single point mutation in the conserved EAL motif (E384A) results in complete loss of the PDE activity of the EAL domain and in a significant decrease in the DGC activity of the GGDEF domain. Structural analyses revealed multiple hydrophobic and aromatic residues around Cys-579 that are necessary for proper DcpA folding and maintenance of the active conformation. On the basis of these observations and taking into account additional bioinformatics analysis of EAL domain–containing proteins, we identified a critical putatively conserved motif, GCxxxQGF that plays an important role in c-di-GMP turnover. We conclude that a substrate-induced conformational switch involving movement of a loop containing a conserved motif in the bifunctional diguanylate cyclase–phosphodiesterase DcpA controls c-di-GMP turnover in M. smegmatis.

The role of ω‐subunit of Escherichia coli RNA polymerase in stress response

Genes to Cells

ppGpp, an alarmone for stringent response, plays an important role in the reprogramming of the transcription complex at the time of stress. In Escherichia coli, ppGpp mediates its action by binding to at least two different sites on RNA polymerase (RNAP). One of the sites to which ppGpp binds to RNAP is at the β′‐ω interface; however, the underlying molecular mechanism and the physiological relevance of ppGpp binding to this site remain unclear. In this study, we have performed UV cross‐linking experiments using 32P azido‐labeled ppGpp to probe its association with RNAP in the absence and presence of ω, and observed weaker binding of ppGpp to the RNAP without ω. Furthermore, we followed the binding kinetics of ppGpp to RNAP with and without ω by isothermal titration calorimetry and found it to be concurrent with the cross‐linking results. Native ω is intrinsically disordered, and we have used a previously characterized structured mutant of ω, which affects the plasticity of the active site of RNAP. Results show that the flexibility conferred by the unstructured ω is a prerequisite for ppGpp binding to RNAP. We have analyzed the stress‐associated phenotypes in an E. coli strain devoid of ω (∆rpoZ). ppGpp levels in ∆rpoZ strain were found to be similar to that of the wild‐type strain. Interestingly, when the ∆rpoZ strain of E. coli was transferred after nutritional stress to an enriched media, the recovery of growth was compromised. We have identified a new phenotype of ∆rpoZ strain corresponding to defect in biofilm formation in minimal media.

Altered Distribution of RNA Polymerase Lacking the Omega Subunit within the Prophages along the Escherichia coli K-12 Genome


The RNA polymerase (RNAP) of Escherichia coli K-12 is a complex enzyme consisting of the core enzyme with the subunit structure α2ββ′ω and one of the σ subunits with promoter recognition properties. The smallest subunit, omega (the rpoZ gene product), participates in subunit assembly by supporting the folding of the largest subunit, β′, but its functional role remains unsolved except for its involvement in ppGpp binding and stringent response. As an initial approach for elucidation of its functional role, we performed in this study ChIP-chip (chromatin immunoprecipitation with microarray technology) analysis of wild-type and rpoZ-defective mutant strains. The altered distribution of RpoZ-defective RNAP was identified mostly within open reading frames, in particular, of the genes inside prophages. For the genes that exhibited increased or decreased distribution of RpoZ-defective RNAP, the level of transcripts increased or decreased, respectively, as detected by reverse transcription-quantitative PCR (qRT-PCR). In parallel, we analyzed, using genomic SELEX (systemic evolution of ligands by exponential enrichment), the distribution of constitutive promoters that are recognized by RNAP RpoD holoenzyme alone and of general silencer H-NS within prophages. Since all 10 prophages in E. coli K-12 carry only a small number of promoters, the altered occupancy of RpoZ-defective RNAP and of transcripts might represent transcription initiated from as-yet-unidentified host promoters. The genes that exhibited transcription enhanced by RpoZ-defective RNAP are located in the regions of low-level H-NS binding. By using phenotype microarray (PM) assay, alterations of some phenotypes were detected for the rpoZ-deleted mutant, indicating the involvement of RpoZ in regulation of some genes. Possible mechanisms of altered distribution of RNAP inside prophages are discussed.

Pup recycling regulates the proteasome.

The FEBS Journal

The Pup proteasome system (PPS) in bacteria is equivalent to the eukaryotic ubiquitin proteasome system (UPS) that allows controlled protein degradation. Unlike the UPS, however, the PPS machinery and regulation is still poorly understood. In this issue of The FEBS Journal, Gur and colleagues combine experimental and modelling analyses to show how the PPS maintains steady-state levels of protein pupylation and consequently tightly controlled protein degradation.