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Masters of Science / Tuberculosis (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a size-reduced subunit β1 (denoted β1m for “minimized”) of Thermus thermophilus RNAP, in which the central domain has been replaced by a Gly-Gly linker, were produced and purified using affinity nickel nitrilotriaceticacid and glutathione-Stransferase (GST) affinity chromatography techniques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.

ExsA is an AraC-family transcriptional regulator that controls expression of T3SS genes in P. aeruginosa. ExsA binds to DNA at T3SS promoters and activates transcription. In the work presented here I examine the stoichiometry, ligand-interaction properties, and transcriptional activation mechanism of ExsA. I determined that ExsA is largely monomeric in solution. ExsA binds T3SS promoter DNA with high affinity resulting in two ExsA-DNA complexes. Whereas the lower molecular weight complex represents a single molecule of ExsA bound to DNA, the higher molecular weight complex represents two molecules of ExsA bound to adjacent sites at T3SS promoters. I next analyzed the mechanism by which ExsD negatively effects ExsA function. Chromatin Immuno-Precipitation Assays (ChIP) demonstrate that ExsD inhibits the DNA-binding activity of ExsA in vivo. Finally, I characterized the mechanism of transcriptional activation by ExsA. ExsA-dependent promoters contain regions that resemble consensus σ70 -35 and -10 recognition hexamers. The spacing between these regions, however, is increased 4-5 bp compared to the σ70 consensus. Nevertheless, I demonstrate that T3SS promoters are dependent on σ70-RNA polymerase (RNAP). Using the abortive initiation assay I discovered that ExsA recruits RNA polymerase to the PexsC and PexsD promoters. Potassium permanganate footprints indicate that following recruitment, RNAP facilitates unwinding of DNA at the -10 hexamer of T3SS promoters. Transcriptional activators generally recruit RNAP by contacting the α or σ70 subunits (or both). I have found that ExsA recruits RNAP to the PexsC and PexsD promoters by contacting region 4.2 of σ70. Although I have established the role of the -10 hexamer, the function of a near-consensus, putative -35 remains puzzling. in vitro transcription assays with mutations in the PexsC -35 hexamer reveals that this region is dispensable for ExsA-independent transcription. This data may suggest that what was thought to be a -35 hexamer is really just an ExsA binding site. Consistent with this hypothesis, I provide evidence that suggests an extended -10 element at PexsC may function to compensate for the lack of a -35 hexamer.

RNA polymerase (RNAP) is the enzyme that catalyzes synthesis of RNA. Mycobacterial RNAP significantly differs from RNAPs from other bacterial species. It requires special transcription factors such as RbpA or CarD. Another difference is the presence of a small RNA (sRNA), Ms1, that binds to mycobacterial RNAP. Ms1 regulates the amount of RNAP in the cell. In our laboratory we recently discovered MoaB2, a new binding partner of mycobacterial A (encoded by sigA), an RNAP subunit, which is essential for recognition of the initial promoter sequence and initiation of transcription. The function of MoaB2 in the regulation of transcription and gene expression is still unknown. The first aim of this Thesis is contribute to elucidation of the mechanism by which Ms1 affects the amount of RNAP. The experiments revealed that this regulation occurs at the level of transcription; Ms1 affects the activity of promoter(s) that drive the transcription of rpoB- rpoC that encode the two catalytic subunits of RNAP. The second aim of this Thesis is to characterize the interactions of MoaB2 with protein of the transcription apparatus. The results confirmed the interaction of MoaB2 with A and showed that neither RNAP nor transcription factors RbpA and CarD are required for this interaction. Finally, a role of the...

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Indiana University-Purdue University Indianapolis (IUPUI) / RNA Polymerase II (Pol II) is the primary actor in the transcription of mRNA from genes. Pol II is a complex composed of twelve protein subunits. This study focused on the changes in the interactome of Rbp3 in S. cerevisiae when the Pol II subunit Rpb9 is removed. Rpb3 is one of the core subunits of Pol II, and any significant changes to the Rpb3 incteractome due to the loss of Rpb9 can be used to infer new information about Rpb9’s role in the Pol II complex. Rpb3 was pulled down using FLAG purification from both wild type and rpb9Δ S. cerevisiae cultures. Rpb3 and the proteins complexed with it were then analyzed using multi-dimensional protein identification technology (MudPIT), a form of liquid chromatography-mass spectrometry (LC-MS). This data was searched using the SEQUEST database search algorithm, and the results were further analyzed for likelihood of interaction using Significance Analysis of INTeractome (SAINT), as well as for post-translational phosphorylation. Deletion of rpb9 did not present any changes in Pol II phosphorylation however it did cause several changes in the interaction network. The rpb9Δ strain showed new interactions with Rtr1, Sen1, Vtc4, Pyc1, Tgl4, Sec61, Tfb2, Hfd1, Erv25, Rib4, Sla1, Ubp15, Bbc1, and Hxk1. The most prominent of these hits are Rtr1, an Rpb1 C-terminal domain phosphatase linked to transcription termination, and Sen1, an RNA/DNA nuclease that terminates transcription. In addition, this mutant showed no interaction with Mtd1, an interaction that is present in the wild type. In all cases, these hits should be considered fuel for future research, rather than conclusive evidence of novel interactions.

Bacteria are the most abundant organisms on the planet. They live almost in all environments, including those that are most extreme. All land and water ecosystems depend heavily upon their activity. Bacteria play essential roles in cycling of nutrients such as carbon, nitrogen, and sulphur. Due to their short cell cycle, they must be able to swiftly adapt to the conditions of their habitat to survive. Microbial growth itself is an autocatalytic process. There are three distinct phases of the growth curve: lag, exponential (log), and stationary. Bacterial cells must change their gene expression between these phases in order to adapt to the new conditions. The first stage of gene expression is transcription. The key enzyme of this stage is RNA polymerase (RNAP) that transcribes DNA into RNA. RNAP is regulated by a number of accessory proteins and also small molecule effectors. Understanding how RNAP functions is essential for understanding how bacteria cope with changing environments. This Thesis presents studies of selected aspects of bacterial gene expression regulation at the level of transcription, using Bacillus subtilis as the model organism. The first part of this Thesis focuses on protein determinants of the ability of RNAP to be regulated by the concentration of the initiating nucleoside...

RNA polymerase (RNAP) is a key multi-subunit enzyme of gene expression that, together with the σ factor, forms a holoenzyme and transcribes genetic information from DNA to RNA. RNAP from Bacillus subtilis and its primary factor σA were studied in this thesis. The σA factor determines the specificity for the promoters to which the holoenzyme binds. Part of its structure is domain 1.1, which is likely to prevent binding of σA to the promoter by itself (unless it is part of the holoenzyme) by binding to domains 2 and 4. The first part of the thesis verifies the hypothesis that domain 1.1 binds domains 2 and 4 and thus prevents binding of σA to the promoter. To this end, various domain constructs have been created and their interactions have been tested. Domain interaction was tested by Nitrocellulose Filter Binding Assay, EMSA, and in vitro transcription. The results did not show significant interaction between domains. The second part of the thesis deals with the creation of a tool for the study of the enzymatology of RNAP from B. subtilis - recombinant RNAP (rRNAP). First, a plasmid construct for expression of rRNAP in Escherichia coli was constructed by a series of cloning steps, followed by protein isolation and characterization. Isolation was achieved without contamination by σ factors (this...

Transcription is a major step in expression of genes of a given organism. Due to environmental constrains this step must be regulated in the favour of the sustenance and growth of the organism. Here comes the relevance of transcription factors, mostly proteins which regulate transcription. One such important group of transcription factors is the zinc finger proteins. It is well known that in eukaryotes the C2H2 zinc finger domain containing proteins are the largest group of transcription factors while in prokaryotes the largest group of transcription factors are represented by helix-turn-helix motif containing proteins. Till now only two C2H2 zinc finger domain proteins-Ros and Muc have been found in alpha proteobacteria which are also transcription factors. In eukaryotes the second largest group of zinc finger proteins have their zinc ion coordinated by four cysteine residues- the C4 zinc finger proteins. They make the nuclear hormone receptor superfamily of proteins. They have also been shown to act as transcription factors. But in eubacteria no such proteins have been described in details except an isolated report of crystal structure of a C-terminal zinc finger domain protein- Jann_2411 from Jannaschia sp. Though a lot of transcription factors have been described in mechanistic details in Escherichia coli and Bacillus subtilis, the list of well described mycobacterial transcription factors is short. Given this fact and the lack of any known zinc finger domain transcription factor in actinobacteria we wanted to see whether M. smegmatis genome also encode any homologue of Jann_2411 and if does whether they have ability to modulate transcription. To meet our aim we did BLASTP search against the genome of M. smegmatis using Jann_2411 as query. We found four C-terminal zinc finger domain proteins –Msmeg_0118. Msmeg_3613, Msmeg_3408 and Msmeg_1531, which we named as Mycobacterial single zinc finger protein (Mszfp) and numbered- Mszfp1, Mszfp2, Mszfp3 and Mszfp4, respectively. Mszfp1 and Mszfp2 were chosen for study as they were the top most hits. In this thesis:- Chapter1 introduces zinc finger proteins, transcription and several levels of control of transcription process in eubacteria. In chapter2 we characterised Mszfp1 biophysically and probed its secondary structure content and oligomeric state in the native and demetallated conditions. We have also shown that this conserved hypothetical protein is expressed throughout the growth phase of M. smegmatis, regulated by SigA and SigB. We have also showed that Mszfp1 is a DNA binding protein in the native state and the demetallated protein has altered DNA binding ability. It was noted that on over expression Mszfp1 affects colony morphology and biofilm forming ability, of M. smegmatis. In chapter3 the ability of Mszfp1 to bind to RNA polymerase of M. smegmatis has been explored. It was found that Mszfp1 can activate transcription by interacting with CTD/NTD of α subunit and domain 4 of σA like CRP on type II CRP activated promoter. In chapter4 similar to Mszfp1 the biophysical study of Mszfp2 has been carried out. It was found that Mszfp2 is also a predominantly alpha helical protein with oligomeric structure having DNA binding ability. Similar to Mszfp1 Mszfp2 on over expression changes the colony morphology. Chapter5 deals with the RNA polymerase binding ability of Mszfp2 and its ability to activate transcription by interacting with CTD/NTD of α subunit but not the σA. In chapter6 we have presented a glimpse of the possible biophysical properties of Mszfp3 and Mszfp4 and given a snapshot of distribution of homologues of Mszfps among other actinobacteria. We have also put forward a hypothesis about the origin of C4 and CCHC zinc finger domains. Chapter7 is the summary of the work embedded in the earlier chapters. In Appendix I is described the making of a bacteria (Bacillus licheniformis) driven heat engine. Appendix II describes an effort to study the visco-elastic properties of Mycobacterium smegmatis cells.

Magister Scientiae - MSc / Tuberculosis (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.