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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

RelZ-Mediated Stress Response in Mycobacterium smegmatis: pGpp Synthesis and Its Regulation

Journal of Bacteriology

Stringent response is a conserved stress response mechanism in which bacteria employ the second messengers guanosine tetraphosphate and guanosine pentaphosphate [collectively termed (p)ppGpp] to reprogram their cellular processes under stress. In mycobacteria, these alarmones govern a multitude of cellular phenotypes, such as cell division, biofilm formation, antibiotic tolerance, and long-term survival. Mycobacterium smegmatis possesses the bifunctional RelMsm as a (p)ppGpp synthetase and hydrolase. In addition, it contains a short alarmone synthetase MS_RHII-RSD (renamed RelZ), which contains an RNase H domain in tandem with the (p)ppGpp synthetase domain. The physiological functions of RelMsm have been well documented, but there is no clear picture about the cellular functions of RelZ in M. smegmatis. RelZ has been implicated in R-loop induced stress response due to its unique domain architecture. In this study, we elucidate the differential substrate utilization pattern of RelZ compared to that of RelMsm. We unveil the ability of RelZ to use GMP as a substrate to synthesize pGpp, thereby expanding the repertoire of second messengers known in mycobacteria. We have demonstrated that the pGpp synthesis activity of RelZ is negatively regulated by RNA and pppGpp. Furthermore, we investigated its role in biofilm formation and antibiotic tolerance. Our findings highlight the complex role played by the RelZ in cellular physiology of M. smegmatis and sheds light upon its functions distinct from those of RelMsm

Unraveling the Role of Silent Mutation in the ω-Subunit of Escherichia coli RNA Polymerase: Structure Transition Inhibits Transcription

ACS Omega

The bacterial RNA polymerase is a multi-subunit enzyme complex composed of six subunits, α2ββ’σω. The function of this enzyme is to transcribe the DNA base sequence to the RNA intermediate, which is ultimately translated to protein. Though the contribution of each subunit in RNA synthesis has been clearly elucidated, the role of the smallest ω-subunit is still unclear despite several studies. Recently, a study on a dominant negative mutant of rpoZ has been reported in which the mutant was shown to render the RNA polymerase defective in transcription initiation (ω6, N60D) and gave an insight on the function of ω in RNA polymerase. Serendipitously, we also obtained a silent mutant, and the mutant was found to be lethal during the isolation of toxic mutants. The primary focus of this study is to understand the mechanistic details of this lethality. Isolated ω shows a predominantly unstructured circular dichroism profile and becomes α-helical in the enzyme complex. This structural transition is perhaps the reason for this lack of function. Subsequently, we generated several silent mutants of ω to investigate the role of codon bias and the effect of rare codons with respect to their position in rpoZ. Not all silent mutations affect the structure. RNA polymerase when reconstituted with structurally altered silent mutants of ω is transcriptionally inactive. The CodonPlus strain, which has surplus tRNA, was used to assess for the rescue of the phenotype in lethal silent mutants.

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

mSystems

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.

Sugar Vinyl Sulfoxide Glycoconjugation of Peptides and Lysozyme: Abrogation of Proteolysis at the Lysine Sites

Biochemistry

We describe a glycoconjugation strategy in which a sugar vinyl sulfoxide, acting as Michael donor, reacts efficiently with amine nucleophiles arising from the lysine side chain in peptides and proteins, at physiological pH and temperature. The method permits glycoconjugation of the lysine residues present in lysozyme with the sugar vinyl sulfoxide. The glycoconjugation of the protein abrogates the trypsin-mediated proteolysis at the lysine sites. The modified protein catalyzes digestion of the Gram-negative Escherichia coli cell wall and retains the same antimicrobial property as the native lysozyme.