Roles of transcription factors, RBPA and SIGF, in the mycobacterium tuberculosis

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