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The vast majority of mechanistic studies on bacterial transcription initiation have used Escherichia coli ( Eco) RNAP as a model.
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Promoter DNA sequences are recognized by holo ( Murakami et al., 2002), triggering a series of conformational changes as the enzyme unwinds 12 to 14 base pairs of DNA to generate the transcription bubble and loads the template-strand (t-strand) DNA into the RNAP active site, resulting in the transcriptionally-competent open promoter complex (RPo Saecker et al., 2011).
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Thus, RNAP is a proven drug target, highlighting the importance of gaining a structural and functional understanding of mycobacterial transcription - an understanding made difficult by the lack of a mycobacterial RNAP structure.īacterial transcription initiation is controlled by promoter-specificity σ-factors, which associate with the core RNAP (α 2ββ’ω subunits), generating the holoenzyme (holo Gruber and Gross, 2003 Murakami and Darst, 2003). RNA polymerase (RNAP), responsible for all transcription in bacteria, is the target of the rifamycin class of antibiotics, a first-line therapeutic treatment for tuberculosis ( Chakraborty and Rhee, 2015). The bacterial pathogen Mycobacterium tuberculosis ( Mtb) is the causative agent of tuberculosis, an ongoing world health problem.
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This work provides a framework to understand the control of mycobacterial transcription by RbpA and CarD. In vivo studies demonstrate the essential role of RbpA, show the effects of RbpA truncations on transcription and cell physiology, and indicate additional functions for RbpA not evident in vitro. Both CarD and RbpA bind near the upstream edge of the −10 element where they likely facilitate DNA bending and impede transcription bubble collapse. We determined a 2.76 Å-resolution crystal structure of a mycobacterial transcription initiation complex (TIC) with RbpA as well as a CarD/RbpA/TIC model. Once RPo forms, CarD also disfavors its isomerization back to RP2. Mechanistic analyses of promoter open complex (RPo) formation establish that RbpA and CarD cooperatively stimulate formation of an intermediate (RP2) leading to RPo formation of RP2 is likely a bottleneck step at the majority of mycobacterial promoters. RbpA and CarD are essential transcription regulators in mycobacteria.