Single-molecule analysis reveals the mechanism of transcription activation in
            <i>M. tuberculosis</i>

Vishwakarma, Rishi K.; Cao, Anne-Marinette; Morichaud, Zakia; Perumal, Ayyappasamy Sudalaiyadum; Margeat, Emmanuel; Brodolin, Konstantin

doi:10.1126/sciadv.aao5498

Cited by 20 publications

(34 citation statements)

References 41 publications

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“…Based on the similarities between the effects of RbpA and of the TGTG motif on transcription, we propose that RbpA could strengthen the interaction of the σ subunit domains 3 and 4 with promoter DNA upstream of the −10 element. This hypothesis is supported by our smFRET study showing that σ B in the Mtb RNAP holoenzyme adopts a conformation incompatible with binding to −10/-35 promoters and that RbpA stabilizes σ B in a conformation compatible with binding ( 27 ). The finding that σ4 was still required for transcription initiation in the context of the extended −10 sin P3 and sig AP-TGTG promoters even in the presence of RbpA suggests that σ4 may have additional roles in initiation, probably in organizing RNAP clamp or β subunit flap domains for RPo formation, as proposed for E. coli σ 70 ( 44 ).…”

Section: Discussionsupporting

confidence: 59%

“…Recently, we demonstrated that RbpA stabilizes the ‘open’ conformation of the σ B subunit in Mtb RNAP. This is optimal for recognition of the −10/-35 promoters, but is dispensable for recognition of the extended −10 promoters ( 27 ). Here, to better understand the molecular basis of this promoter specificity, we explored the effect of mutations in σ B and RbpA on Mtb RNAP activity at promoter variants that harbor different combinations of the extended −10 and −35 motifs.…”

Section: Introductionmentioning

confidence: 99%

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RbpA relaxes promoter selectivity of M. tuberculosis RNA polymerase

Perumal

Vishwakarma

et al. 2018

Nucleic Acids Research

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The transcriptional activator RbpA associates with Mycobacterium tuberculosis RNA polymerase (MtbRNAP) during transcription initiation, and stimulates formation of the MtbRNAP-promoter open complex (RPo). Here, we explored the influence of promoter motifs on RbpA-mediated activation of MtbRNAP containing the stress-response σB subunit. We show that both the ‘extended −10’ promoter motif (T-17G-16T-15G-14) and RbpA stabilized RPo and allowed promoter opening at suboptimal temperatures. Furthermore, in the presence of the T-17G-16T-15G-14 motif, RbpA was dispensable for RNA synthesis initiation, while exerting a stabilization effect on RPo. On the other hand, RbpA compensated for the lack of sequence-specific interactions of domains 3 and 4 of σB with the extended −10 and the −35 motifs, respectively. Mutations of the positively charged residues K73, K74 and R79 in RbpA basic linker (BL) had little effect on RPo formation, but affected MtbRNAP capacity for de novo transcription initiation. We propose that RbpA stimulates transcription by strengthening the non-specific interaction of the σ subunit with promoter DNA upstream of the −10 element, and by indirectly optimizing MtbRNAP interaction with initiation substrates. Consequently, RbpA renders MtbRNAP promiscuous in promoter selection, thus compensating for the weak conservation of the −35 motif in mycobacteria.

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Section: Discussionsupporting

confidence: 59%

Section: Introductionmentioning

confidence: 99%

RbpA relaxes promoter selectivity of M. tuberculosis RNA polymerase

Perumal

Vishwakarma

et al. 2018

Nucleic Acids Research

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“…To determine whether the s subunit anti-pausing activity can be observed with RNAP from other bacteria, we studied initial transcription by MtbRNAP. We used the M. tuberculosis s B subunit that requires the activator protein RbpA to stabilize its active conformation in the MtbRNAP holoenzyme (Vishwakarma et al, 2018). As RbpA N-terminus binds within the RNA-exit channel, it could modulate s4.2 anti-pausing activity (model in Fig.…”

Section: Rbpa From M Tuberculosis Stimulates Translocation Through Tmentioning

confidence: 99%

“…Binding of the regions s3.2 and s4.1 within the RNA exit channel takes place during assembly of the RNAP holoenzyme when the s subunit undergoes the transition from the "closed" to the "open" conformation (Callaci et al, 1999). Recent single-molecule fluorescence resonance energy transfer studies demonstrated that in Mycobacterium tuberculosis, this transition is regulated by the activator protein RbpA (Vishwakarma et al, 2018) that interacts with the s2 and s3.2 domains (Boyaci et al, 2018). Whether RbpA can influence initial transcription has never been explored.…”

Section: Introductionmentioning

confidence: 99%

Anti-pausing activity of region 4 of the RNA polymerase σ subunit and its regulation by σ-remodeling factors

Brodolin

Morichaud

2020

Preprint

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The basal transcription factors of cellular RNA polymerases (RNAPs) stimulate the initial RNA synthesis via poorly understood mechanisms. Here, we explored the mechanism employed by the bacterial factor σ in promoter-independent initial transcription. We found that the RNAP holoenzyme lacking the promoter-binding domain σ4 is ineffective in de novo transcription initiation and displays high propensity to pausing upon extension of RNAs 3 to 7 nucleotides in length. The σ4 domain stabilizes short RNA:DNA hybrids and suppresses pausing by stimulating RNAP active-center translocation. The anti-pausing activity of σ4 is modulated by its interaction with the β subunit flap domain and by the σ remodeling factors AsiA and RbpA. Our results suggest that the presence of σ4 within the RNA exit channel compensates for the intrinsic instability of short RNA:DNA hybrids by increasing RNAP processivity, thus favoring productive transcription initiation. This ‘RNAP boosting’ activity of the initiation factor is shaped by the the thermodynamics of RNA:DNA interactions and thus, should be relevant for any factor-dependent RNAP.

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“…Data from structural and biophysical studies suggest that free group 1 and 2 σ subunits adopt a "closed" inactive conformation in which the spatial arrangement of domains σ2 and σ4 is incompatible with promoter DNA binding. Binding to core RNAP induces or stabilizes an "open, " active σ conformation, optimal for promoter binding (Callaci et al, 1999;Schwartz et al, 2008;Vishwakarma et al, 2018). Canonically, RNAP activity at promoters is regulated through DNA-binding transcription factors (Browning and Busby, 2016) that recognize and bind to specific motifs on dsDNA (DB-TFs) and influence the initiation pathway steps after promoter binding ( Figure 1A).…”

Section: Introductionmentioning

confidence: 99%

The σ Subunit-Remodeling Factors: An Emerging Paradigms of Transcription Regulation

Vishwakarma

Brodolin

2020

Front. Microbiol.

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Transcription initiation is a key checkpoint and highly regulated step of gene expression. The sigma (σ) subunit of RNA polymerase (RNAP) controls all transcription initiation steps, from recognition of the −10/−35 promoter elements, upon formation of the closed promoter complex (RPc), to stabilization of the open promoter complex (RPo) and stimulation of the primary steps in RNA synthesis. The canonical mechanism to regulate σ activity upon transcription initiation relies on activators that recognize specific DNA motifs and recruit RNAP to promoters. This mini-review describes an emerging group of transcriptional regulators that form a complex with σ or/and RNAP prior to promoter binding, remodel the σ subunit conformation, and thus modify RNAP activity. Such strategy is widely used by bacteriophages to appropriate the host RNAP. Recent findings on RNAP-binding protein A (RbpA) from Mycobacterium tuberculosis and Crl from Escherichia coli suggest that activator-driven changes in σ conformation can be a widespread regulatory mechanism in bacteria.

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Single-molecule analysis reveals the mechanism of transcription activation in M. tuberculosis

Abstract: Mycobacterial activator RbpA stimulates transcription by stabilizing open conformation of the RNA polymerase σ subunit.

Cited by 20 publications

References 41 publications

RbpA relaxes promoter selectivity of M. tuberculosis RNA polymerase

RbpA relaxes promoter selectivity of M. tuberculosis RNA polymerase

Anti-pausing activity of region 4 of the RNA polymerase σ subunit and its regulation by σ-remodeling factors

The σ Subunit-Remodeling Factors: An Emerging Paradigms of Transcription Regulation

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