Mycobacterium RbpA cooperates with the stress-response σB subunit of RNA polymerase in promoter DNA unwinding

Abstract

RbpA, a transcriptional activator that is essential for Mycobacterium tuberculosis replication and survival during antibiotic treatment, binds to RNA polymerase (RNAP) in the absence of promoter DNA. It has been hypothesized that RbpA stimulates housekeeping gene expression by promoting assembly of the σA subunit with core RNAP. Here, using a purified in vitro transcription system of M. tuberculosis, we show that RbpA functions in a promoter-dependent manner as a companion of RNAP essential for promoter DNA unwinding and formation of the catalytically active open promoter complex (RPo). Screening for RbpA activity using a full panel of the M. tuberculosis σ subunits demonstrated that RbpA targets σA and stress-response σB, but not the alternative σ subunits from the groups 3 and 4. In contrast to σA, the σB subunit activity displayed stringent dependency upon RbpA. These results suggest that RbpA-dependent control of RPo formation provides a mechanism for tuning gene expression during the switch between different physiological states, and in the stress response.

Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA

“…1C), confirming previous sequencebased structural predictions (15)(16)(17). Four residues of 15-residue RbpA-BL connecting the RbpA-SID to the RCD are also visible in the structure.…”

“…Adding favorable protein/DNA contacts to the transcription initiation complex would potentially stabilize a transcription initiation intermediate, which is consistent with findings that RbpA activates transcription primarily by stimulating RPo formation (15,17). The role of RbpA-R79, which structural modeling predicts could contact the nt-strand DNA phosphate backbone at the −14 position (Fig.…”

“…The RbpA-σ 2 interaction is mediated by the C-terminal segment [which we designate here the σ-interaction domain (SID)], and point mutations that disrupt σ-binding also disrupt RbpA function (15). In addition to primary σ-factors, RbpA interacts with certain group 2 σ-factors (σ B in Mtb and σ HrdA in Sco) but does not interact with group 3 or 4 σ-factors (15)(16)(17). RbpA is present at transcription Significance Initiation of transcription in bacteria relies on a multisubunit RNA polymerase in concert with a dissociable σ-subunit that confers promoter recognition and opening to reveal the DNA template strand.…”

Mycobacterium tuberculosis Transcription Machinery: Ready To Respond to Host Attacks

“…Additional contacts between RbpA and RNAP ␤ have been proposed based on cross-linking experiments (63, 73,74), but the recent structural modeling of RbpA onto an RNAP-promoter open complex would be incompatible with these interactions (71), suggesting that further analysis will be needed to resolve these inconsistencies. RbpA has been shown to increase the affinity of the factor to the core RNAP, increase the affinity of RNAP holoenzyme to promoter DNA, and facilitate the formation of RP o (71,75,76), all of which could contribute to the ability of RbpA to promote RNAP-promoter complex formation and stability. The housekeeping factor A has been reported to have an affinity for M. tuberculosis RNAP core enzyme similar to that of the alternative factor F (74), in which case RbpA may be necessary to improve A affinity and competitiveness for RNAP under conditions that require the activity of A .…”

Early diagnosis and effective treatment regimens are the keys to tackle antimicrobial resistance in tuberculosis (TB): A report from Euroscicon's international TB Summit 2016

“…The initiation factors are positioned near the upstream edge of the transcription bubble. 43,44 Techniques such as fluorescent labeling were used to study the open complex using M. bovis RNAP which is known to form open complexes that are more unstable as compared to E.coli RNAP. It was found that the initiation factors act independently and cooperatively to regulate transcription and that only in the presence of both factors does the transcription kinetics in mycobacteria appear to be similar to that observed in E. coli.…”