Domains within RbpA Serve Specific Functional Roles That Regulate the Expression of Distinct Mycobacterial Gene Subsets

Abstract

The RNA polymerase (RNAP) binding protein A (RbpA) contributes to the formation of stable RNAP-promoter open complexes (RP) and is essential for viability in mycobacteria. Four domains have been identified in the RbpA protein, i.e., an N-terminal tail (NTT) that interacts with RNAP β' and σ subunits, a core domain (CD) that contacts the RNAP β' subunit, a basic linker (BL) that binds DNA, and a σ-interaction domain (SID) that binds group I and group II σ factors. Limited studies have been performed in mycobacteria, however, and how individual structural domains of RbpA contribute to RbpA function and mycobacterial gene expression remains mostly unknown. We investigated the roles of the RbpA structural domains in mycobacteria using a panel of mutants that target individual RbpA domains. The function of each RbpA domain was required for viability and optimal growth in We determined that the RbpA SID is both necessary and sufficient for RbpA interaction with the RNAP, indicating that the primary functions of the NTT and CD are not solely association with the RNAP. We show that the RbpA BL and SID are required for RP stabilization , while the NTT and CD antagonize this activity. Finally, RNA-sequencing analyses suggest that the NTT and CD broadly activate gene expression, whereas the BL and SID activate or repress gene expression in a gene-dependent manner for a subset of mycobacterial genes. Our findings highlight specific outcomes for the activities of the individual functional domains in RbpA. is the causative agent of tuberculosis and continues to be the most lethal infectious disease worldwide. Improved molecular understanding of the essential proteins involved in transcription, such as RbpA, could provide targets for much needed future therapeutic agents aimed at combatting this pathogen. In this study, we expand our understanding of RbpA by identifying the RbpA structural domains responsible for the interaction of RbpA with the RNAP and the effects of RbpA on transcription initiation and gene expression. These experiments expand our knowledge of RbpA while also broadening our understanding of bacterial transcription in general.

CarD contributes to diverse gene expression outcomes throughout the genome of Mycobacterium tuberculosis

“…In addition to cis-acting PRR sequences, transacting factors, such as other transcription factors, could also contribute to the effect of CarD on transcription of a particular gene. Mtb encodes another essential RNAP-interacting protein, RbpA (39,40), which is also capable of stabilizing RP o formed by mycobacterial RNAP in vitro (41)(42)(43). Structural studies have shown that association of CarD and RbpA with the same RNAP holoenzyme is feasible (43,44), and we have shown that CarD and RbpA can function cooperatively to stabilize RP o (41).…”

The calculation of transcript flux ratios reveals single regulatory mechanisms capable of activation and repression

“…. [14] Since the rate of transcript production is merely the probability of a promoter occupying C times the irreversible rate out (k 5 ), we have the steady-state rate of transcript production as A k open k close + k escape [16] or ϕ = k escape k open k on ½R k ce k combined , [17] where k ce = k close + k escape [18] and k combined = k on ½R + k off + k open − k open ðk close − k on ½RÞ k close + k escape . [19] This expression for ϕ is completely consistent with previous derivations of the steady-state rate of transcription initiation for the same mechanism (23): The calculation of the control coefficient for each parameter was determined using MATLAB's symbolic math toolbox and leads to the following equations: [28] The flux control coefficients obtained provide a quantification of how strongly the flux depends on each parameter.…”

“…For example, the lambda repressor can either stimulate RP o formation by interacting with the polymerase or repress transcription by competing with RNAP-promoter interactions depending on the precise position of its operator sites relative to the promoter (9)(10)(11). An alternative strategy can be seen in the case of transcription factors that are recruited to the initiation complex by a protein-protein interaction with the RNAP directly [e.g., CarD (12,13), RbpA (14), and DskA (15)]. Here, promoter sequence cannot be directly read out via a DNA binding domain, and the relative physical arrangement of the factor and RNAP cannot be modulated.…”

“…Here, promoter sequence cannot be directly read out via a DNA binding domain, and the relative physical arrangement of the factor and RNAP cannot be modulated. However, these factors are still capable of producing promoter-specific regulatory outcomes as exemplified by DksA (15) and RbpA (14). In this case, differential regulation must be driven by an indirect effect of DNA sequence on the kinetics of transcription initiation.…”

CarD and RbpA modify the kinetics of initial transcription and slow promoter escape of the Mycobacterium tuberculosis RNA polymerase

“…Separating the CD and SID is a basic linker (BL) region that interacts with promoter DNA just upstream of the −10 element (15,19,29,30,35) (Figure 1B and C). While both the BL and SID are necessary to promote a stable RP o in vitro , removal of the NTT and the CD leads to enhanced RP o stability relative to wild-type RbpA (25). However, in vivo RNA-sequencing analyses have indicated that the NTT and CD are responsible for a wide-ranging activation of gene expression, whereas the BL and SID can either activate or repress transcription depending on the promoter (25).…”