Academic literature on the topic 'Mycobacterium tuberculosis SigK'

Abstract Mycobacterium tuberculosis (MTB) is the causative of tuberculosis, a disease, which causes 2 millions of death every year, with a dramatic incidence especially in developing countries. To find new drug and vaccine strategies against MTB, it is of fundamental importance to study the mechanisms, that allow its survival to environmental stresses, to which it is subjected during the period of infection and latency in the host macrophages. The fine transcriptional regulation of specific genes in response to stress conditions and the peculiar structure of its wall play a key role on this. In the first part of the PhD project two mycobacterial sigma factors, SigE and SigF, which regulate the transcription of specific genes in response to various environmental stresses such as surface stress, oxidative stress, alkaline pH and thermal shock, have been characterized. First the transcriptional regulation, translational and post-translational regulation of the extracytoplasmic function (ECF) transcription factor SigE were studied. Regarding the study of the transcriptional regulation, it was possible to confirm by 5'RACE PCR and RT-PCR experiments the presence of three promoters of sigE, and to determine the contribution of each promoter in the transcription of this gene, depending on the environmental conditions of bacterial growth. The fact, that the transcriptional start codon of one of these promoters is located 63 base pairs downstream of the start codon annotated in MTB genome opened the possibility of the existence of two isoforms of Sige. By translational fusions between specific sequences of sigE with lacZ, deprived of its own translational initiation codon, and subsequent site-specific mutagenesis, it was possible to confirm, based on further beta-galactosidase activity detection, the existence of two alternative start codons, an ATC and a TTG, coding for an isoform of respectively 218 and 215 of amino acids, in addition to the ATG already annotated in MTB genome, which encodes for an isoform of 257 amino acids. Finally, it was possible to confirm, that the gene downstream sigE encodes for the anti-sigma factor of SigE, called RseA, capable of binding both isoforms of SigE. In a second project also the role of the factor SigF M. smegmatis in the biosynthesis of carotenoid pigments, resistance to hydrogen peroxide and in the efficiency of bacterial transformation was studied. By RT-PCR it has been shown, that SigF controls the transcription of genes involved in the biosynthesis of carotenoid pigments, and, assuming that they serve as protection against free radicals, it was verified that the sigF mutant strain is actually more sensitive compared to the wild type strain to treatment with hydrogen peroxide. Finally, we also demonstrated, that the mutant strain has a higher transformation efficiency than the wild type strain, indicating that SigF regulates the transcription of genes possibly involved in the permeability of the cell wall. In the second part of the project, the localization of the protein on the surface PPE17 mycobacteria was characterized. Like other members of the PPE family, the PPE17 has a highly conserved N-terminal domain, which, based on different evidences in literature, is assumed to play an important role in their translocation to the mycobacterial surface. Moreover, it was investigated the possible influence of the presence of PE11 in the translocation process or in the stability of PPE17, as the PE11 coding sequence is in tandem and co-transcribed with that encoding the PPE17, and there is a specific interaction between these two proteins. The data obtained by proteinase K sensitivity assays performed on M. smegmatis strains, expressing the entire PPE17 or only its domain PPE (dPPE17) fused with the HA epitope, confirm, that the entire PPE17 is exposed on the surface, both in the presence and absence of PE11. According to data obtained, the possibility to translocate the MTB model antigen (Mpt64) on the surface of the vaccine strain M. bovis BCG, by fusing them with the dPPE17 was tested. Proteinase K and whole cell ELISA assays performed on cultures of M. bovis BCG expressing this chimeric protein indicate, that it is indeed localized at the mycobacterial surface. Similarly, another two fusions with dPPE17 were constructed to express on the mycobacterial surface the multimeric MTB antigen AG85-ESAT6 of MTB and the Csp C3 antigen of Plasmodium bergii. According to the proteinase K sensitivity assays carried out on strains of M. smegmatis expressing the two chimeric proteins indicate that also in this case both are localized at the surface. The strains of M. bovis BCG expressing these antigens on their surface will be tested in future in the mouse model to measure any increase in protection compared to the wild type strain.<br>Riassunto Mycobacterium tuberculosis (MTB) è l’agente eziologico della tubercolosi, patologia che nel mondo causa ogni anno due milioni di morti, con un’incidenza drammatica specie nei Paesi in via di sviluppo. Per poter trovare nuove strategie farmacologiche e vaccinali contro MTB è di fondamentale importanza lo studio dei meccanismi, che permettono la sua sopravvivenza ai vari stress ambientali, ai quali è sottoposto durante il periodo di infezione e latenza nei macrofagi dell’ospite. La fine regolazione della trascrizione di geni specifici in risposta a condizioni di stress e la peculiare struttura della sua parete giocano in merito un ruolo fondamentale. Nella prima parte del progetto di dottorato sono stati caratterizzati due fattori di trascrizione sigma micobatterici, SigE e SigF, che regolano la trascrizione di geni specifici in risposta a vari tipi di stress ambientali, come lo stress di superficie, lo stress ossidativo, il pH alcalino e lo shock termico. Anzitutto è stata studiata la regolazione trascrizionale, traduzionale e posttraduzionale del fattore di trascrizione con funzione extracitoplasmatica (ECF) SigE. Per quanto riguarda lo studio della regolazione trascrizionale, è stato possibile confermare tramite esperimenti di 5’RACE PCR e RT-PCR la presenza di tre promotori di sigE, e a dosare, a seconda delle condizioni ambientali di crescita batterica, il contributo di ciascun promotore nella trascrizione di questo gene. Dato che l’inizio della trascrizione di uno di questi promotori è sito 63 paia di basi a valle del codone di start annotato nel genoma, si è aperta l’ipotesi dell’esistenza di due isoforme di SigE. Mediante fusioni traduzionali tra specifiche sequenze di sigE con lacZ, private del proprio codone di inizio della traduzione, e successive mutagenesi sito-specifiche, è stato possibile confermare, in base all’attività beta-galattosidasica rilevata, l’esistenza di due codoni di start alternativi, un ATC ed un TTG, che codificano per un’isoforma di rispettivamente 218 e 215 di amminoacidi, oltre all’ATG già annotato nel genoma di MTB, che codifica per un’isoforma di 257 amminoacidi. Infine è stato possibile confermare, che il gene a valle di sigE codifica per il fattore anti-sigma di SigE, denominato RseA, in grado di legare entrambe le isoforme di SigE. In un secondo progetto è stato studiato anche il ruolo del fattore SigF di M. smegmatis nella biosintesi di pigmenti carotenoidi, nella resistenza a perossido d’idrogeno e nell’efficienza di trasformazione batterica. Tramite RT-PCR è stato dimostrato che SigF controlla la trascrizione di geni coinvolti nella biosintesi dei pigmenti carotenoidi, e, partendo dal presupposto che essi fungono da protezione contro i radicali liberi, è stato verificato che il mutante per il gene sigF è effettivamente più sensibile rispetto al ceppo selvatico al trattamento con perossido d’idrogeno. Infine è stato dimostrato anche, che il ceppo mutante possiede una maggiore efficienza di trasformazione rispetto al ceppo selvatico, indicando che SigF regola probabilmente la trascrizione di geni coinvolti nella permeabilità della parete. Nella seconda parte del progetto è stata caratterizzata la localizzazione della proteina PPE17 sulla superficie micobatterica. Come altri membri della famiglia PPE, la PPE17 presenta un dominio N-terminale altamente conservato, il quale, in base a diverse evidenze in letteratura, si suppone svolgere un ruolo importante per la loro traslocazione in superficie. Inoltre, si è voluto verificare un’eventuale influenza della presenza della proteina PE11 nel processo di traslocazione in o nella stabilità della PPE17, in quanto la sequenza codificante la PE11 è in tandem e co-trascritta con quella codificante la PPE17, e vi è un’interazione specifica tra queste due proteine. I dati ottenuti mediante saggi di sensibilità alla proteinasi K su ceppi di M. smegmatis, esprimenti la PPE17 intera o solo il suo dominio PPE (dPPE17) fuse all’epitopo HA, confermano che la PPE17 intera sia esposta in superficie, sia in presenza che in assenza di PE11. In base ai dati ottenuti si è infine tentato di veicolare un antigene modello (Mpt64) di MTB sulla superficie del ceppo vaccinale M. bovis BCG fondendolo con il dPPE17. Saggi di sensibilità alla proteinasi K e ELISA su cellule intere effettuati su culture di M. bovis BCG esprimenti questa proteina chimerica indicano, che essa sia effettivamente localizzata a livello superficiale. Allo stesso modo sono state costruite due ulteriori fusioni con il dPPE17 per esprimere sulla superficie micobatterica l’antigene multimerico Ag85-ESAT6 di MTB e l’antigene Csp C3 di Plasmodium berghii. In base a saggi di sensibilità alla proteinasi K svolti su ceppi di M. smegmatis esprimenti le due fusioni anche in questo caso entrambe localizzano in superficie. I ceppi di M. bovis BCG esprimenti questi antigeni sulla loro superficie saranno testati in futuro nel modello del topo per misurare un eventuale aumento della protezione rispetto al ceppo selvatico.

Mycobacterium tuberculosis is the causative agent of Tuberculosis (TB). Two billion people are currently infected with M. tuberculosis bacilli, one in ten of whom will develop active TB in their lifetime. M. tuberculosis is able to survive within macrophages but the exact mechanisms used for intracellular survival are poorly understood. DNA-dependent RNA polymerase is the enzyme responsible for transcription in all living organisms. In bacteria this enzyme recognises different promoters by binding to sigma factors that recognise those promoters. This study focuses on the role and regulation of the M. tuberculosis extracytoplasmic function (ECF) sigma factor, SigG. ECF sigma factors are responsible for upregulating genes necessary for bacterial stress responses. SigG has previously been shown to be upregulated in response to DNA damage and during macrophage infection. It has been demonstrated that sigG is expressed from at least 2 promoters and that only promoter P1 is DNA-damage inducible. sigG is co-transcribed with the two downstream genes Rv0181c and Rv0180c, which were hypothesised to be anti- and anti-anti-sigma factors to SigG. Protein-protein interaction studies showed that SigG and Rv0181c do not interact. Potential anti-sigma factors to SigG were identified, the most promising of which was the thioredoxin family protein Rv1084. Two potential SigG-dependent genes had previously been identified, Rv0887c and Rv0911. It has been demonstrated that SigG is able to bind to the promoter regions of these genes but this interaction was not specific. An M. tuberculosis sigG-Rv0180c deletion strain was constructed and complemented with the whole operon as well as with sigG alone. The phenotype of the mutant strain was examined in vitro as well as in vivo to test the hypothesis that SigG has a role during infection. Use of a phenotype microarray revealed an enhanced susceptibility of the mutant strain to oleic acid and its ester, Tween 80, leading to investigation of the sensitivity of the strains to a range of fatty acids.

La tuberculose est causée par l'infection par Mycobacterium tuberculosis (M. Tb). Les interactions entre M. Tb et les récepteurs de l'immunité innée participent à la mise en place de la et au développement de la pathologie tuberculeuse. DC-SIGN est une lectine de type C qui joue un rôle majeur dans l'infection par M. Tb. Afin de mieux comprendre sa fonction dans l'immunité anti-tuberculeuse, nous avons étudié son rôle dans le modèle murin d'infection par M. Tb. Parmi les 8 homologues murins (SIGNR1-8), SIGNR1 et SIGNR3 sont les formes les plus conservées par rapport à DC-SIGN. L'utilisation de souris inactivées dans les gènes codant pour SIGNR1-3 et 5 nous a permis de montrer que seules les souris SIGNR3-/- présentent une susceptibilité accrue à M. Tb. SIGNR3, est induite au cours de l'infection à la surface de phagocytes pulmonaires, et peut interagir avec M. Tb ou des composés glycosylés mycobactériens pour permettre leur internalisation et l'induction d'effecteurs de la réponse immunitaire. In vitro, l'activation de SIGNR3 induit une voie de signalisation dépendante d'un motif intracellulaire et de la kinase Syk qui active une cascade de transduction pour moduler la réponse inflammatoire. Nos résultats suggèrent que SIGNR3 est l'homologue fonctionnel de DC-SIGN et contribue à la protection de l'hôte contre l'infection. Chez l'homme l'induction de DC-SIGN par M. Tb semble, elle aussi, induire des cytokines inflammatoires ce qui soulèvent clairement la question de savoir si DC-SIGN doit être encore considéré comme un récepteur permettant l'échappement de M. Tb à la réponse immunitaire ou plutôt comme un récepteur impliqué dans la protection de l'hôte<br>Tuberculosis (TB) is due to the infection by Mycobacterium tuberculosis (M. Tb). The interaction between the bacilli and the pathogen recognition receptors regulate the innate immune response to the pathogen but can also contribute to the development of the pathology. DC-SIGN is a C-type lectin which interacts with M. Tb may play an important role in human TB, which remained to be further investigated. To better understand the role of DC-SIGN in anti-mycobacterial immunity, we have used the murine model of M. Tb infection. Among the eight murine homologues (SIGNR1 to 8), SIGNR1 and SIGNR3 are the most similar homologues to DC-SIGN The infection of the mouse lines inactivated in those 2 candidates and SIGNR5 have shown that only SIGNR3-deficient mice display a higher susceptibility to the early phases of infection. Like DC-SIGN, SIGNR3 is induced in pulmonary phagocytes in infected animals. SIGNR3 can recognize glycosylated mycobacterial ligands and the whole bacteria to allow their internalization and to induce the secretion of effectors of the immune response. In vitro, we have shown that SIGNR3 signaling is dependent on a tyrosine motif and relies on the kinase SYK which activates the immune-modulatory transduction cascade. Our results suggest that SIGNR3 is the functional homologue of DC-SIGN and contributes to the protection of the host. Preliminary results suggest that DC-SIGN stimulation may also promote the secretion of pro-inflammatory cytokines in human macrophages. In summary, our results challenge the current dogma on the role of DC-SIGN as an immune escape receptor, and rather suggest that DC-SIGN may be a key-component of the defense system against M. Tb and possibly other pathogens, which remains to be evaluated in human

Mycobacterium tuberculosis is a remarkably successful human pathogen, which causes nearly two million death annually. Although tuberculosis has been widely studied for more than a century, the mechanisms by which it causes the disease, are poorly understood. Recent progress of molecular genetics beside of completion of its genome sequence, have been providing tools for new approaches to a better understanding of M.tuberculosis biochemistry, physiology and pathogenesis. Here we show a series of studies in which, taking advantage of a genetic approach, we characterized different aspects of M. tuberculosis physiology which might have a strong impact in drug discovery and in the understanding of its virulence mechanism. Producing a knock-down mutant of sigA, encoding the principal sigma factor of M. tuberculosis, we showed that it is essential and posed the basis for the development of a simple in vitro test to select molecules active on dormant bacteria. Similar results were obtained using .a genetic system in which the expression of different toxins belonging to toxin-antitoxin modules can be induced. We also produced an M. tuberculosis mutant in which the gene encoding the alternative sigma factor SigE and its cognate anti-sigma factor RseA were deleted. The SigE-RseA regulatory network is extremely important to stop phagosome maturation and consequently for virulence. This mutant strain will be complemented with the sigE-rseA couple carrying different mutations that will allow the further characterization of this regulatory network. Finally, we also characterized the regulation of the expression of the PE_PGRS30 structural gene and the role of its PGRS domain in virulence. Keywords: M.tuberculosis, sigA, sigE, Toxin-antitoxin, PE_PGRS<br>Mycobacterium tuberculosis is a remarkably successful human pathogen, which causes nearly two million death annually. Although tuberculosis has been widely studied for more than a century, the mechanisms by which it causes the disease, are poorly understood. Recent progress of molecular genetics beside of completion of its genome sequence, have been providing tools for new approaches to a better understanding of M.tuberculosis biochemistry, physiology and pathogenesis. Here we show a series of studies in which, taking advantage of a genetic approach, we characterized different aspects of M. tuberculosis physiology which might have a strong impact in drug discovery and in the understanding of its virulence mechanism. Producing a knock-down mutant of sigA, encoding the principal sigma factor of M. tuberculosis, we showed that it is essential and posed the basis for the development of a simple in vitro test to select molecules active on dormant bacteria. Similar results were obtained using .a genetic system in which the expression of different toxins belonging to toxin-antitoxin modules can be induced. We also produced an M. tuberculosis mutant in which the gene encoding the alternative sigma factor SigE and its cognate anti-sigma factor RseA were deleted. The SigE-RseA regulatory network is extremely important to stop phagosome maturation and consequently for virulence. This mutant strain will be complemented with the sigE-rseA couple carrying different mutations that will allow the further characterization of this regulatory network. Finally, we also characterized the regulation of the expression of the PE_PGRS30 structural gene and the role of its PGRS domain in virulence. Keywords: M.tuberculosis, sigA, sigE, Toxin-antitoxin, PE_PGRS

M. Tuberculosis se lie aux cellules dendritiques par l'intermédiaire d'une lectine de type C (DC-SIGN). Des études antérieures ont suggéré que DC-SIGN puisse différencier une espèce mycobactérienne pathogène M. Tuberculosis d'une espèce non pathogène M. Smegmatis. Ce phénomène semblait être attribué à une structure particulière des coiffes mannosylées du lipoarabinomannane (ManLAM). Durant ma thèse, nous avons montré que DC-SIGN est capable de différencier les espèces appartenant au complexe tuberculosis des autres espèces. Cependant, nos résultats indiquent que la discrimination par DC-SIGN n'est pas due à une structure particulière des coiffes ou à la localisation du ManLAM, et que d'autres ligands pourraient intervenir dans la liaison, telles que les glycoprotéines. Ainsi, la liaison entre DC-SIGN et les espèces du complexe tuberculosis semble plus compliqué que se que l'on avait imaginé et résulterait de la liaison coopérative de plusieurs ligands<br>M. Tuberculosis binds to dendritic cells through a C-type lectin (DC-SIGN: dendritic cell-specific ICAM-3 Grabbing Non integrin). Previous studies suggested that DC-SIGN differentially binds to the pathogenic M. Tuberculosis and the non-pathogenic M. Smegmatis. This feature has been tentatively attributed to differencies in the LAM cap structures. During my thesis, we enlarged this finding by showing that DC-SIGN is able to discriminate M. Tuberculosis complex species from others species whatever their pathogenic status. Furthermore, we showed that this differential binding cannot be associated to LAM cap structures or localization, but rather to the presence of other potential ligands including glycoproteins. Thus the binding between DC-SIGN and the M. Tuberculosis complex species appears to de more complicated then previously suspected and seems to be due to cooperative interaction of DC-SIGN with several ligands

A distinctive feature of host-pathogen interactions in the case of Mycobacterium tuberculosis is the asymptomatic latent phase of infection. The ability of the bacillus to survive for extended periods of time in the host suggests an adaptive mechanism in M. tuberculosis that can cope with a variety of environmental stresses and other host stimuli. Extensive genomic studies and analysis of knock-out phenotypes revealed elaborate cellular machinery in M. tuberculosis that ensures a rapid cellular response to host stimuli. Prominent amongst these are two-component systems and σ factors that exclusively govern transcription re-engineering in response to environmental stimuli. M. tuberculosis σK is a σ factor that was demonstrated to control the expression of secreted antigenic proteins. The study reported in this thesis was geared to understand the molecular basis for σK activity as well as to explore conditions that would regulate σK activity. Transcription in bacteria is driven by the RNA polymerase enzyme that can associate with multiple σ factors. σ factors confer promoter specificity and thus directly control the expression of genes. The association of different σ factors with the RNA polymerase is essential for the temporal and conditional re-engineering of the expression profile. Environment induced changes in expression rely on a subset of σ factors. This class of σ factors (also referred to as Class IV or Extra-cytoplasmic function (ECF) σ factors) is regulated by a variety of mechanisms. The regulation of an ECF σ factor activity at the transcriptional, translational or posttranslational steps ensures fidelity in the cellular concentration of free, active ECF σ factors. In general, ECF σ factors associate with an inhibitory protein referred to as an anti-σ factor. The release of a free, active σ factor from a σ /anti-σ complex is thus a mechanism that can potentially control the cellular levels of an active σ factor in the cell. M. tuberculosis σK is associated with a membrane bound anti-σK (also referred to as RskA) (Said-Salim et al., Molecular Microbiology 62: 1251-1263: 2006). The extracellular stimulus that is recognized by RskA remains unclear. However, recent studies have suggested the possibility of a regulated proteolytic cascade that can selectively degrade RskA and other membrane associated anti-σ factors. The goal of the study was to understand this regulatory mechanism with a specific focus on the M. tuberculosis σK/RskA complex. The structure of the cytosolic σK/RskA complex and the associated biochemical and biophysical characteristics revealed several features of this /anti-σ complex that were hitherto unclear. In particular, these studies revealed a redox sensitive regulatory mechanism in addition to a regulated proteolytic cascade. These features and an analysis of the M. tuberculosis σK/RskA complex vis-à-vis the other characterized σ/anti σfactor complexes are presented in this thesis. This thesis is organized as follows- Chapter 1 provides an overview of prokaryotic transcription. A brief description of the physiology of M. tuberculosis is presented along with a summary of characterized factors that contribute to the pathogenecity and virulence of this bacillus. The pertinent mechanistic issues of σ/anti-σ factor interactions are placed in the context of environment mediated changes in M. tuberculosis transcription. A summary of studies in this area provides a background of the research leading to this thesis. Chapters 2 and 3 of this thesis describe the structural and mechanistic studies on the σK/RskA complex. The crystal structure of the σK/RskA complex revealed a disulfide bond in domain 4 (σK4). σK4 interacts with the -35 element of the promoter DNA. The disulfide forming cysteines were seen to be conserved in more than 70% of σK homologs, across both gram-positive and gram-negative bacteria. The conservation of the disulfide-forming cysteines led us to further characterize the role of this disulfide in σK/RskA interactions. These were examined by several biochemical and biophysical experiments. The redox potential of these disulfide bond forming cysteine residues were consistent with the proposed role of a sensor. The crystal structure and biochemical studies thus suggest that M. tuberculosis σK is activated under reducing conditions. Chapter 4 of this thesis describes the progress made thus far in the structural and biochemical characterization of an intra-membrane protease, M. tuberculosis Rip1 (Rv2869c). This protein is an essential component of the proteolytic cascade that selectively cleaves RskA. The proteolytic steps that govern the selective degradation of an anti-σ factor were first characterized in the case of E. coli σE (Li, X. et al. Proc. Natl. Acad. Sci. USA, 106:14837-14842, 2009). This cascade is triggered by the concerted action of a secreted protease (also referred to as a site-1 protease) and a trans-membrane protease (also referred to as a site-2 protease). M. tuberculosis Rip1 was demonstrated to be bona-fide site 2 protease that acts on three anti-σ factors viz., RskA, RslA and RsmA (Sklar et al., Molecular Microbiology 77:605-617; 2010). To further characterize the role of Rip1 in the proteolytic cascade, this intra-membrane protease was cloned, expressed and purified for structural, biochemical and biophysical analysis. The preliminary data on this membrane protein is described in this chapter. The conclusions from the studies reported in this thesis and the scope for future work in this area is described in Chapter 5. Put together, the σK/RskA complex revealed facets of σ/anti-σ factor interactions that were hitherto unrecognized. The most prominent amongst these is the finding that an ECF σfactor can respond to multiple environmental stimuli. Furthermore, as seen in the case of the σK/RskA complex, the σ factor can itself serve as a receptor for redox stimuli. Although speculative, a hypothesis that needs further study is whether these features of the σK/RskA complex contribute to the variable efficacy of the M. bovis BCG vaccine. In this context it is worth noting that σK governs the expression of the prominent secreted antigens- MPT70 and MPT83. The studies reported in this thesis thus suggest several avenues for future research to understand mycobacterial diversity, immunogenicity and features of host-pathogen interactions. The appendix section is divided into two subparts- Appendix 1 of the thesis is a review on peptidase V. This is a chapter in The Handbook of Proteolytic enzymes (Elsevier Press, ISBN:9780123822192). Appendix 2 of the thesis includes technical details and an extended materials and methods section.

The mechanism of prokaryotic transcription has been characterized primarily in the classic system, Escherichia coli, and cannot be confidently extended to include other prokaryotic species, such as those of the Actinobacteria phylum. Actinobacteria represents a diverse group of Gram-positive species that range from soil dwellers to obligate pathogens, such as Mycobacterium tuberculosis (Tb). These species encode RNA polymerase (RNAP) binding proteins that are not present in model organisms, and therefore present a unique lens through which the basic mechanism of transcription can be further explored outside of model systems. In addition, these mechanisms of transcriptional regulation can be studied in the context of M. Tuberculosis pathogenesis. The model we use for tuberculosis is Mycobacterium Smegmatis, a homologue, which has a faster doubling time and is only Biosafety level 1. Within Actinobacteria, notable conserved RNAP binding proteins include RNA polymerase binding protein A (RbpA) and CarD. RbpA is specific to Actinobacteria, binding the β subunit of RNAP and primary σ factors. CarD binds to the β subunit and associates with DNA. Both proteins are upregulated upon exposure to stress, and have implications in the initiation of rRNA transcription. Each is proposed to stimulate the formation of transcriptionally competent RNAP-holoenzyme open promoter complexes, and CarD is thought to act as a global transcriptional regulator. RbpA and CarD are believed to be essential in M. Tuberculosis and M. Smegmatis. Recent structural analyses of RbpA and CarD suggest the two proteins may share a region of similarity that could compete for binding to the β subunit, and brings into question whether the two proteins are capable of coordinately modulating transcription or antagonize each other's activity. This was investigated through purification of CarD and RbpA and in vitro studies performed with [α-32P] Uridine triphosphate used to measure the level of transcription. These experiments led to the conclusion that RbpA and CarD are able to associate with the same RNAP and have an additive stabilizing action on the polymerase. Whether or not RbpA is an essential protein was also investigated genetically, and by using a Tetracycline on/off system. Sigma factors play an important role in transcription due to their ability to recognize promoter regions and initiate transcription. One connection that we have preliminary data for, through DNA pull downs, is that sigF binds rRNA promoters, and CarD and RbpA are both studied in the context of rRNA transcription. Therefore sigF is another factor that could be regulating rRNA transcription, possibly during stress. SigF is also the sigma factor that responds to oxidative stress, and CarD is involved in oxidative stress. Sigma F is a member of a family of 13 different sigma factors that are preset in M. Tuberculosis. There are two different types of sigma factors: primary, which are essential for normal growth, and alternative, which are typically expressed during differing environmental conditions. Sigma F has been shown to be upregulated during oxidative stress, which is why it was of particular interest to us. To investigate the roles of sig F, we exposed sig F deletion mutants and wild type strains to oxidative stress and measured ribosomal RNA production by reverse transcription quantitative real time PCR. It was concluded that sigF is a probable suppressor of rRNA when exposed to oxidative stress.