Investigating the functional interaction of transcription regulator CarD of Mycobacterium tuberculosis with Ribonucleic Acid Polymerase

dc.contributor.advisorSchubert, Wolf-Dieter
dc.contributor.authorMapotsane, Thuso
dc.date.accessioned2015-05-29T09:42:55Z
dc.date.accessioned2024-05-09T07:45:21Z
dc.date.available2015-05-29T09:42:55Z
dc.date.available2024-05-09T07:45:21Z
dc.date.issued2014
dc.descriptionMasters of Scienceen_US
dc.description.abstractTuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). TB mainly affects lungs of patients but other parts of the body can also be affected. It kills approximately 2 million people annually. HIV/AIDS and drug resistance make TB difficult to control. Mtb CarD protein forms a physiological complex with Ribonucleic Acid Polymerase (RNAP). This complex causes Mtb to undergo dormancy rendering it difficult to control using current antibiotics. CarD and a size-reduced subunit β1 (denoted β1m for “minimized”) of Thermus thermophilus RNAP, in which the central domain has been replaced by a Gly-Gly linker, were produced and purified using affinity nickel nitrilotriaceticacid and glutathione-Stransferase (GST) affinity chromatography techniques respectively. CarD N terminal domain (CarDN) was generated from CarD by inserting a stop codon by site directed mutagenesis. CarD was stabilised by adding 5 % (v/v) glycerol to PBS pH 7.4 ensuring protein stability of up to 67 days rather than 2 days without glycerol. CarDN was stable in PBS pH 7.4 without addition of glycerol. This suggests that the CarD C terminal domain may be responsible for CarD instability. To further purify the proteins both anion exchange and gel permeation chromatography techniques were used. CarD and CarDN degrade immediately after anion exchange potentially because of the high ion concentration which partially unfolds the protein making it prone to proteolytic cleavage. GST-pull down assays were used to demonstrate complex formation between RNAP β1m and both CarD and CarDN confirming that complex formation is dependent on the N-terminal domain of CarD.en_US
dc.identifier.urihttps://hdl.handle.net/10566/13321
dc.language.isoenen_US
dc.publisherUniversity of the Western Capeen_US
dc.rights.holderUniversity of the Western Capeen_US
dc.subjectTuberculosisen_US
dc.subjectCarD/RNAP complexen_US
dc.subjectMycobacterium tuberculosisen_US
dc.titleInvestigating the functional interaction of transcription regulator CarD of Mycobacterium tuberculosis with Ribonucleic Acid Polymeraseen_US
dc.typeThesisen_US

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