https://libjncir.jncasr.ac.in/xmlui/handle/10572/16 Sat, 04 Apr 2026 05:27:33 GMT 2026-04-04T05:27:33Z https://libjncir.jncasr.ac.in/xmlui/handle/10572/2602 Mycobacterium tuberculosis Rho Is an NTPase with Distinct Kinetic Properties and a Novel RNA-Binding Subdomain Mitra, Anirban; Misquitta, Rachel; Nagaraja, V. Two mechanisms - factor independent and dependent termination - ensure the completion of RNA synthesis in eubacteria. Factor-dependent mechanism relies on the Rho protein to terminate transcription by interacting with RNA polymerase. Although well studied in Escherichia coli, the properties of the Rho homologs from most bacteria are not known. The rho gene is unusually large in genus Mycobacterium and other members of actinobacteria, having,150 additional residues towards the amino terminal end. We describe the distinct properties of Rho from Mycobacterium tuberculosis. It is an NTPase with a preference for purine nucleoside triphosphates with kinetic properties different from E. coli homolog and an ability to use various RNA substrates. The N-terminal subdomain of MtbRho can bind to RNA by itself, and appears to contribute to the interaction of the termination factor with RNAs. Furthermore, the interaction with RNA induces changes in conformation and oligomerization of MtbRho. Open Access Wed, 01 Jan 2014 00:00:00 GMT https://libjncir.jncasr.ac.in/xmlui/handle/10572/2602 2014-01-01T00:00:00Z https://libjncir.jncasr.ac.in/xmlui/handle/10572/2597 Conditional silencing of topoisomerase I gene of Mycobacterium tuberculosis validates its essentiality for cell survival Ahmed, Wareed; Menon, Shruti; Godbole, Adwait Anand; Karthik, Pullela V. D. N. B.; Nagaraja, V. Topoisomerases are an important class of enzymes for regulating the DNA transaction processes. Mycobacterium tuberculosis (Mtb) is one of the most formidable pathogens also posing serious challenges for therapeutic interventions. The organism contains only one type IA topoisomerase (Rv3646c), offering an opportunity to test its potential as a candidate drug target. To validate the essentiality of M.tuberculosis topoisomerase I (TopoI(Mt)) for bacterial growth and survival, we have generated a conditionally regulated strain of topoI in Mtb. The conditional knockdown mutant exhibited delayed growth on agar plate. In liquid culture, the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the M.tuberculosis growth and open up new avenues for targeting the enzyme. Restricted Access Wed, 01 Jan 2014 00:00:00 GMT https://libjncir.jncasr.ac.in/xmlui/handle/10572/2597 2014-01-01T00:00:00Z https://libjncir.jncasr.ac.in/xmlui/handle/10572/2598 Direct regulation of topoisomerase activity by a nucleoid-associated protein Ghosh, Soumitra; Mallick, Bratati; Nagaraja, V. The topological homeostasis of bacterial chromosomes is maintained by the balance between compaction and the topological organization of genomes. Two classes of proteins play major roles in chromosome organization: the nucleoid-associated proteins (NAPs) and topoisomerases. The NAPs bind DNA to compact the chromosome, whereas topoisomerases catalytically remove or introduce supercoils into the genome. We demonstrate that HU, a major NAP of Mycobacterium tuberculosis specifically stimulates the DNA relaxation ability of mycobacterial topoisomerase I (TopoI) at lower concentrations but interferes at higher concentrations. A direct physical interaction between M. tuberculosis HU (MtHU) and TopoI is necessary for enhancing enzyme activity both in vitro and in vivo. The interaction is between the amino terminal domain of MtHU and the carboxyl terminal domain of TopoI. Binding of MtHU did not affect the two catalytic trans-esterification steps but enhanced the DNA strand passage, requisite for the completion of DNA relaxation, a new mechanism for the regulation of topoisomerase activity. An interaction-deficient mutant of MtHU was compromised in enhancing the strand passage activity. The species-specific physical and functional cooperation between MtHU and TopoI may be the key to achieve the DNA relaxation levels needed to maintain the optimal superhelical density of mycobacterial genomes. Open Access Wed, 01 Jan 2014 00:00:00 GMT https://libjncir.jncasr.ac.in/xmlui/handle/10572/2598 2014-01-01T00:00:00Z https://libjncir.jncasr.ac.in/xmlui/handle/10572/2600 Molecular Basis for the Differential Quinolone Susceptibility of Mycobacterial DNA Gyrase Kumar, Rupesh; Madhumathi, Bhavani Shankar; Nagaraja, V. DNA gyrase is a type II topoisomerase that catalyzes the introduction of negative supercoils in the genomes of eubacteria. Fluoroquinolones (FQs), successful as drugs clinically, target the enzyme to trap the gyrase-DNA complex, leading to the accumulation of double-strand breaks in the genome. Mycobacteria are less susceptible to commonly used FQs. However, an 8-methoxy-substituted FQ, moxifloxacin (MFX), is a potent antimycobacterial, and a higher susceptibility of mycobacterial gyrase to MFX has been demonstrated. Although several models explain the mechanism of FQ action and gyrase-DNA-FQ interaction, the basis for the differential susceptibility of mycobacterial gyrase to various FQs is not understood. We have addressed the basis of the differential susceptibility of the gyrase and revisited the mode of action of FQs. We demonstrate that FQs bind both Escherichia coli and Mycobacterium tuberculosis gyrases in the absence of DNA and that the addition of DNA enhances the drug binding. The FQs bind primarily to the GyrA subunit of mycobacterial gyrase, while in E. coli holoenzyme is the target. The binding of MFX to GyrA of M. tuberculosis correlates with its effectiveness as a better inhibitor of the enzyme and its efficacy in cell killing. Restricted Access Wed, 01 Jan 2014 00:00:00 GMT https://libjncir.jncasr.ac.in/xmlui/handle/10572/2600 2014-01-01T00:00:00Z