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This study elucidates several mechanisms of action associated with the GTPase protein CgtA from Mycobacterium smegmatis mc2 155. The first chapter of this thesis presents findings from biochemical studies aimed at exploring the effects of truncation mutation in CgtAms. The presence of 70S and 50S, along with an excess of GTP in the reaction, leads to an increase in specific GTPase activity. The activity of GTPase rises with the deletion of the C-terminal domain in CgtAms during the reaction. The binding assay of ribosome (70S and 50S) proteins (CgtAms and C-terminal domain deleted CgtA) conducted through ultracentrifugation density gradient indicates that the C-terminal domain of CgtAms enhances the interaction with CgtAms and the ribosome. The results indicate that the two domains of CgtAms, namely the Obg domain and the GTPase domain, interact with each other, allowing for signal transmission from the Obg domain to the GTPase domain or the other way around. In the second chapter four mutant strains have been successfully created from the wild-type strain of M. smegmatis: C-terminus merodiploid (CTD cgtA+ CTD cgtA::hygr), C-terminus knock out (ΔCTD cgtA::hygr), cgtA merodiploid (cgtA+cgtA::hygr), and cgtA knock out (ΔcgtA::hygr). The growth curve of mutant strains shows a significant decrease compared to the wild-type strain. The size, width, and area of mutants, including C-terminus merodiploid (CTD cgtA+ CTD cgtA::hygr), C-terminus knock out (ΔCTD cgtA::hygr), cgtA merodiploid (cgtA+cgtA::hygr), and cgtA knock out (ΔcgtA::hygr), are significantly reduced when compared to wild type cells. The reduction of biofilm formation in cells occurs in 7H10 agar medium following the knockout of cgtA and C-terminus cgtA gene strains. Biofilm formation of the full-length cgtA and C-terminus cgtA gene knockout strains in broth medium does not occur. The third chapter illustrates the role of cgtA, primarily focused on the interaction between a DNA fragment and gDNA through chip technology. The full length of the cgtA ORF and its truncation mutations, specifically the C-terminal deleted cgtA, have been successfully cloned into the pMnyT-kan plasmid vector. Following the successful cloning of the cgtA gene and the creation of truncation mutations, the proteins were expressed, and their expression was confirmed through western blot analysis. The validation of DNA-protein interactions has been accomplished by employing ChiP, followed by the isolation and sequencing of the interacting DNA. Analysis of isolated DNA reveals five anticipated motifs and a coordinated ChiP binding network.
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