Structural basis of the mycobacterial stress-response RNA polymerase auto-inhibition via oligomerization.

Self-assembly of macromolecules into higher-order symmetric structures is fundamental for the regulation of biological processes. Higher-order symmetric structure self-assembly by the gene expression machinery, such as bacterial DNA-dependent RNA polymerase (RNAP), has never been reported before. Here, we show that the stress-response σB factor from the human pathogen, Mycobacterium tuberculosis, induces the RNAP holoenzyme oligomerization into a supramolecular complex composed of eight RNAP units. Cryo-electron microscopy revealed a pseudo-symmetric structure of the RNAP octamer in which RNAP protomers are captured in an auto-inhibited state and display an open-clamp conformation. The structure shows that σB is sequestered by the RNAP flap and clamp domains. The transcriptional activator RbpA prevented octamer formation by promoting the initiation-competent RNAP conformation. Our results reveal that a non-conserved region of σ is an allosteric controller of transcription initiation and demonstrate how basal transcription factors can regulate gene expression by modulating the RNAP holoenzyme assembly and hibernation.

Modular Imaging Scaffold for Single-Particle Electron Microscopy.

Technological breakthroughs in electron microscopy (EM) have made it possible to solve structures of biological macromolecular complexes and to raise novel challenges, specifically related to sample preparation and heterogeneous macromolecular assemblies such as DNA-protein, protein-protein, and membrane protein assemblies. Here, we built a V-shaped DNA origami as a scaffolding molecular system to template proteins at user-defined positions in space. This template positions macromolecular assemblies of various sizes, juxtaposes combinations of biomolecules into complex arrangements, isolates biomolecules in their active state, and stabilizes membrane proteins in solution. In addition, the design can be engineered to tune DNA mechanical properties by exerting a controlled piconewton (pN) force on the molecular system and thus adapted to characterize mechanosensitive proteins. The binding site can also be specifically customized to accommodate the protein of interest, either interacting spontaneously with DNA or through directed chemical conjugation, increasing the range of potential targets for single-particle EM investigation. We assessed the applicability for five different proteins. Finally, as a proof of principle, we used RNAP protein to validate the approach and to explore the compatibility of the template with cryo-EM sample preparation.

Region 4 of the RNA polymerase σ subunit counteracts pausing during initial transcription.

All cellular genetic information is transcribed into RNA by multisubunit RNA polymerases (RNAPs). The basal transcription initiation factors of cellular 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 nucleotide at the RNA 3' end determines the pause lifetime. The σ4 domain stabilizes short RNA:DNA hybrids and suppresses pausing by stimulating RNAP active-center translocation. The antipausing activity of σ4 is modulated by its interaction with the ß 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 thermodynamics of RNA:DNA interactions and thus, should be relevant for any factor-dependent RNAP.

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

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.

RbpA relaxes promoter selectivity of M. tuberculosis RNA polymerase.

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.

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

The σ subunit of bacterial RNA polymerase (RNAP) controls recognition of the -10 and -35 promoter elements during transcription initiation. Free σ adopts a "closed," or inactive, conformation incompatible with promoter binding. The conventional two-state model of σ activation proposes that binding to core RNAP induces formation of an "open," active, σ conformation, which is optimal for promoter recognition. Using single-molecule Förster resonance energy transfer, we demonstrate that vegetative-type σ subunits exist in open and closed states even after binding to the RNAP core. As an extreme case, RNAP from Mycobacterium tuberculosis preferentially retains σ in the closed conformation, which is converted to the open conformation only upon binding by the activator protein RbpA and interaction with promoter DNA. These findings reveal that the conformational dynamics of the σ subunit in the RNAP holoenzyme is a target for regulation by transcription factors and plays a critical role in promoter recognition.

Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria.

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. It has been recently shown that initial RNA synthesis by the bacterial RNA polymerase (RNAP) is interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Using single-molecule FRET and biochemical analysis, here we show that the pause encountered by RNAP after the synthesis of a 6-nt RNA (ITC6) renders the promoter escape strongly dependent on the NTP concentration. Mechanistically, the paused ITC6 acts as a checkpoint that directs RNAP to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway. The cyclic unscrunching/scrunching of the promoter generates a long-lived, RNA-bound paused state; the abortive RNA release and DNA unscrunching are thus not as tightly linked as previously thought. Finally, our new model couples the pausing with the abortive and productive outcomes of initial transcription.

Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes.

Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia coli transcription open complexes. Our results revealed the existence of two stable states within RNAP-DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.

RNA Polymerase Pausing during Initial Transcription.

In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.

Regions 1.2 and 3.2 of the RNA Polymerase σ Subunit Promote DNA Melting and Attenuate Action of the Antibiotic Lipiarmycin.

Initiation of RNA synthesis by bacterial RNA polymerase (RNAP) requires melting of promoter DNA, which is nucleated by the σ subunit during formation of the "open" promoter complex (RPo). The antibiotic lipiarmycin (Lpm) inhibits promoter melting by blocking access of the template DNA strand to the RNAP active-site cleft. Here we show that Escherichia coli RNAP holoenzymes containing either housekeeping σ(70), with a deletion in the region 3.2, or the stationary phase σ(S) subunits exhibited hypersensitivity to Lpm and increased cold sensitivity of RPo formation. Similar effects were produced by mutation located ~60 Å away from the Lpm binding site within σ(70) region 1.2, controlling -10 promoter element recognition. Our data suggested that template strand single-stranded DNA competes with Lpm for binding to RNAP and that σ(70) regions 1.2 and 3.2 attenuate Lpm action by promoting DNA duplex opening.

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

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 σ(A) 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 σ(A) and stress-response σ(B), but not the alternative σ subunits from the groups 3 and 4. In contrast to σ(A), the σ(B) 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.

An innovative biologic recycling process of leukoreduction filters to produce active human antimicrobial peptides.

BACKGROUND: Human neutrophil peptides (HNPs) 1 to 3 are the major antimicrobial peptides of the azurophilic granules of neutrophils. They represent an important arm of the innate immune system. Their production by chemical synthesis and recombinant technologies is expensive and limited by technical constraints due to their composition and the presence of three disulfide bonds. STUDY DESIGN AND METHODS: We have developed an original approach based on the purification of the natural human defensins HNPs 1 to 3 from neutrophils trapped on leukoreduction filters used in blood processing. The purification of HNPs 1 to 3 from these filters is performed in two steps: extraction of HNPs 1 to 3 retained in the filters followed by their immunoprecipitation. Studies were performed to determine the stability of defensins in the filters stored at room temperature. The activity of HNPs 1 to 3 obtained by our rapid protocol was validated by determining minimal inhibitory concentrations (MICs) against six reference bacterial strains and 12 clinical isolates. RESULTS: The human defensins HNPs 1 to 3 extracted from leukoreduction filters displayed high antimicrobial activity against tested strains, with MIC values between 0.12 and 1 µg/mL. Kinetics assays showed the appearance of activity 15 minutes after peptide addition. Moreover, we found that the HNPs 1 to 3 purified from leukoreduction filters that had been stored for 45 days at room temperature remained active. CONCLUSION: Leukoreduction filters provide a rich and safe source of active human defensins HNPs 1 to 3. Moreover, the stability of the peptides in filters stored at room temperature allows envisaging a large-scale development of the process.

Mycobacterium tuberculosis RbpA protein is a new type of transcriptional activator that stabilizes the σ A-containing RNA polymerase holoenzyme.

RbpA is an RNA polymerase (RNAP)-binding protein whose presence increases the tolerance levels of Mycobacteria to the first-line anti-tuberculosis drug rifampicin by an unknown mechanism. Here, we show that the role of Mycobacterium tuberculosis RbpA in resistance is indirect because it does not affect the sensitivity of RNAP to rifampicin while it stimulates transcription controlled by the housekeeping σ(A)-factor. The transcription regulated by the stress-related σ(F) was not affected by RbpA. The binding site of RbpA maps to the RNAP ß subunit Sandwich-Barrel Hybrid Motif, which has not previously been described as an activator target and does not overlap the rifampicin binding site. Our data suggest that RbpA modifies the structure of the core RNAP, increases its affinity for σ(A) and facilitates the assembly of the transcriptionally competent promoter complexes. We propose that RbpA is an essential partner which advantages σ(A) competitiveness for core RNAP binding with respect to the alternative σ factors. The RbpA-driven stimulation of the housekeeping gene expression may help Mycobacteria to tolerate high rifampicin levels and to adapt to the stress conditions during infection.

Structural transitions in the transcription elongation complexes of bacterial RNA polymerase during σ-dependent pausing.

A transcription initiation factor, the σ(70) subunit of Escherichia coli RNA polymerase (RNAP) induces transcription pausing through the binding to a promoter-like pause-inducing sequence in the DNA template during transcription elongation. Here, we investigated the mechanism of σ-dependent pausing using reconstituted transcription elongation complexes which allowed highly efficient and precisely controlled pause formation. We demonstrated that, following engagement of the σ subunit to the pause site, RNAP continues RNA synthesis leading to formation of stressed elongation complexes, in which the nascent RNA remains resistant to Gre-induced cleavage while the transcription bubble and RNAP footprint on the DNA template extend in downstream direction, likely accompanied by DNA scrunching. The stressed complexes can then either break σ-mediated contacts and continue elongation or isomerize to a backtracked conformation. Suppressing of the RNAP backtracking decreases pausing and increases productive elongation. On the contrary, core RNAP mutations that impair RNAP interactions with the downstream part of the DNA template stimulate pausing, presumably by destabilizing the stressed complexes. We propose that interplay between DNA scrunching and RNAP backtracking may have an essential role in transcription pausing and its regulation in various systems.

Antibiotics trapping transcription initiation intermediates: To melt or to bend, what's first?

Promoter DNA melting, culminating in the loading of the single-stranded DNA template into the RNA polymerase active site, is a key step in transcription initiation. Recently, the first transcription inhibitors found to block distinct steps of promoter melting were characterized. Here, the impact of these studies is discussed with respect to the current models of transcription initiation.

The transcription inhibitor lipiarmycin blocks DNA fitting into the RNA polymerase catalytic site.

Worldwide spreading of drug-resistant pathogens makes mechanistic understanding of antibiotic action an urgent task. The macrocyclic antibiotic lipiarmycin (Lpm), which is under development for clinical use, inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Using genetic and biochemical approaches, we show that Lpm targets the sigma(70) subunit region 3.2 and the RNAP beta' subunit switch-2 element, which controls the clamping of promoter DNA in the RNAP active-site cleft. Lpm abolishes isomerization of the 'closed'-promoter complex to the transcriptionally competent 'open' complex and blocks sigma(70)-stimulated RNA synthesis on promoter-less DNA templates. Lpm activity decreases when the template DNA strand is stabilized at the active site through the interaction of RNAP with the nascent RNA chain. Template DNA-strand fitting into the RNAP active-site cleft directed by the beta' subunit switch-2 element and the sigma(70) subunit region 3.2 is essential for promoter melting and for de novo initiation of RNA synthesis, and our results suggest that Lpm impedes this process.

Resistance to rifampicin: at the crossroads between ecological, genomic and medical concerns.

The first antibiotic of the ansamycin family, rifampicin (RIF), was isolated in 1959 and was introduced into therapy in 1962; it is still a first-line agent in the treatment of diseases such as tuberculosis, leprosy and various biofilm-related infections. The antimicrobial activity of RIF is due to its inhibition of bacterial RNA polymerase (RNAP). Most frequently, bacteria become resistant to RIF through mutation of the target; however, this mechanism is not unique. Other mechanisms of resistance have been reported, such as duplication of the target, action of RNAP-binding proteins, modification of RIF and modification of cell permeability. We suggest that several of these alternative resistance strategies could reflect the ecological function of RIF, such as autoregulation and/or signalling to surrounding microorganisms. Very often, resistance mechanisms found in the clinic have an environmental origin. One may ask whether the introduction of the RIF analogues rifaximin, rifalazil, rifapentine and rifabutin in the therapeutic arsenal, together with the diversification of the pathologies treated by these molecules, will diversify the resistance mechanisms of human pathogens against ansamycins.

Myxopyronin: a punch in the jaws of bacterial RNA polymerase.

Evaluation of: Belogurov GA, Vassylyeva MN, Sevostyanova A et al.: Transcription inactivation through local refolding of the RNA polymerase structure. Nature 457, 332-335 (2008) and, Mukhopadhyay J, Das K, Ismail S et al.: The RNA polymerase 'switch region' is a target for inhibitors. Cell 135, 295-307 (2008). Bacterial RNA polymerase is an essential enzyme, which is responsible for synthesizing RNA from a DNA template and is targeted by a number of antibiotics. The mechanism of action of two closely related transcription inhibitors, myxopyronin B and a synthetic analog desmethyl-myxopyronin was elucidated, together with the structures of the antibiotic-RNA polymerase complexes. The studies reveal a new binding site and a new mechanism of action affecting the jaw domain of the enzyme. As the need for new antibiotics increase, these studies open new ways to the synthesis of more potent myxopyronin analogs.

In Xenopus egg extracts, DNA replication initiates preferentially at or near asymmetric AT sequences.

Previous observations led to the conclusion that in Xenopus eggs and during early development, DNA replication initiates at regular intervals but with no apparent sequence specificity. Conversely, here, we present evidence for site-specific DNA replication origins in Xenopus egg extracts. Using lambda DNA, we show that DNA replication origins are activated in clusters in regions that contain closely spaced adenine or thymine asymmetric tracks used as preferential initiation sites. In agreement with these data, AT-rich asymmetric sequences added as competitors preferentially recruit origin recognition complexes and inhibit sperm chromatin replication by increasing interorigin spacing. We also show that the assembly of a transcription complex favors origin activity at the corresponding site without necessarily eliminating the other origins. Thus, although Xenopus eggs have the ability to replicate any kind of DNA, AT-rich domains or transcription factors favor the selection of DNA replication origins without increasing the overall efficiency of DNA synthesis. These results suggest that asymmetric AT-rich regions might be default elements that favor the selection of a DNA replication origin in a transcriptionally silent complex, whereas other epigenetic elements linked to the organization of domains for transcription may have further evolved over this basal layer of regulation.