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Three mechanisms of transcription termination. (A) Intrinsic termination is driven by formation of an RNA hairpin in the emerging transcript, the base of which occurs 8-9 nt from the site of release. Release also requires a uridine-rich segment downstream of the hairpin, particularly in the region immediately adjacent to the GC-rich end of the stem. Although not illustrated, we suggest (as described in the text) that the DNA bubble is partly rewound and that the RNADNA hybrid is partly unwound when the hairpin is fully formed. (B) The termination factor Rho is a hexameric RNA translocase that binds 60 nt of emerging transcript, moving along it in a 5-3 direction in an ATP-dependent reaction. This movement is believed to extract the transcript. (C) Mfd is a DNA translocase that binds duplex DNA upstream of the transcription bubble and RNAP in a region near the site of DNA rewinding. The activity of the translocase causes dissociation of the complex in conditions that do not allow the RNA chain to advance through NTP polymerization.

Three mechanisms of transcription termination. (A) Intrinsic termination is driven by formation of an RNA hairpin in the emerging transcript, the base of which occurs 8-9 nt from the site of release. Release also requires a uridine-rich segment downstream of the hairpin, particularly in the region immediately adjacent to the GC-rich end of the stem. Although not illustrated, we suggest (as described in the text) that the DNA bubble is partly rewound and that the RNADNA hybrid is partly unwound when the hairpin is fully formed. (B) The termination factor Rho is a hexameric RNA translocase that binds 60 nt of emerging transcript, moving along it in a 5-3 direction in an ATP-dependent reaction. This movement is believed to extract the transcript. (C) Mfd is a DNA translocase that binds duplex DNA upstream of the transcription bubble and RNAP in a region near the site of DNA rewinding. The activity of the translocase causes dissociation of the complex in conditions that do not allow the RNA chain to advance through NTP polymerization.

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Article
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By using DNA heteroduplexes that inhibit rewinding of the upstream part of the transcription bubble, we show that transcript release in termination by the enzymes Mfd and Rho is facilitated by reannealing of DNA in the upstream region of the transcription bubble, as is also true for termination by intrinsic terminators. We also show that, like Mfd,...

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Context 1
... that interact with RNAP (1), (ii) the termination factor Rho, an RNA- dependent ATPase and RNA ''helicase'' (or, more accurately, RNA translocase) that acts by binding the emerging transcript and (presumably) RNAP (2,3), and (iii) Mfd, an ATP- dependent DNA translocase that acts on RNAP and DNA upstream of the transcription bubble (4-6) ( Fig. 1). [The rep- lication fork apparatus could contain a fourth mechanism that removes obstructing transcription complexes (7).] Understand- ing these termination pathways may reveal important aspects of transcription complex stability, as well as the nature of regulation that acts through ...
Context 2
... segments that prevent base-pairing in the transcription bubble or in the duplex region upstream of the bubble where Mfd is believed to bind. Nontemplate strand substitutions were used because the base composition of the template strand is constrained by the requirement to stop transcription complexes at the same site by nucleotide starvation. Fig. 1C shows a portion of the template and the presumed nucleic acid structure of the transcription complex at this site; the entire transcript up to position 74 consists of A and C, so that RNA synthesis with only ATP and CTP produces a complex with a defined 74-nt transcript. The depicted size of the transcription bubble fits experimental ...

Citations

... For the RNA release, which defines termination of transcription, the catch-up mode generally assumes a critical role in ρ′s motor activity. The mechanical force exerted by ρ is expected to shear RNA off from RNA·DNA hybrid in the RNA shearing model or displace RNAP forward out of the ribonucleotide (NTP) incorporation site in the RNAP hyper-translocation model 14 . The stand-by mode, on the other hand, generally assumes that RNA is released or EC is disassembled not by the motor action of ρ but by allosteric changes in EC conformation triggered by ρ 7,9,12 . ...
... In the RNA shearing model 14 , ρ pulls RNA to resolve RNA·DNA hybrid for terminational release of RNA and immediate or delayed dissociation of RNAP from DNA. In the RNAP hyper-translocation model 14 , by contrast, ρ pushes RNAP forward on DNA for the collapse of transcription bubble leading to RNA release with simultaneous or subsequent RNAP dissociation. ...
... All open squares are located below the guideline, except for rho terminator slightly above the line. Error bar represents the standard deviation of the mean from n in the nontemplate strand of template DNA at varying positions 14 . If a mismatch overlaps with a transcription bubble region that is not base-paired before termination but becomes base-paired after hyper-translocation for termination, the mismatch would inhibit rewinding to diminish hyper-translocation proficiency but little affect RNA shearing, which does not need rewinding of the region 14,31 . ...
Article
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Rho is a general transcription termination factor in bacteria, but many aspects of its mechanism of action are unclear. Diverse models have been proposed for the initial interaction between the RNA polymerase (RNAP) and Rho (catch-up and stand-by pre-terminational models); for the terminational release of the RNA transcript (RNA shearing, RNAP hyper-translocation or displacing, and allosteric models); and for the post-terminational outcome (whether the RNAP dissociates or remains bound to the DNA). Here, we use single-molecule fluorescence assays to study those three steps in transcription termination mediated by E. coli Rho. We find that different mechanisms previously proposed for each step co-exist, but apparently occur on various timescales and tend to lead to specific outcomes. Our results indicate that three kinetically distinct routes take place: (1) the catch-up mode leads first to RNA shearing for RNAP recycling on DNA, and (2) later to RNAP displacement for decomposition of the transcriptional complex; (3) the last termination usually follows the stand-by mode with displacing for decomposing. This three-route model would help reconcile current controversies on the mechanisms. Rho is a general transcription termination factor in bacteria. Here, Song et al. use single-molecule fluorescence assays to provide evidence that Rho-mediated transcription termination can occur via three kinetically different routes.
... To further test this model, we used exonuclease III (Exo III) footprinting of the upstream DNA border 37 and found that the tDNA in the que-ePEC indeed primarily resides in a post-translocated register ( Figure S8A). Notably, the hyper-translocation of the RNA, but not the DNA, causes the hybrid to be weakened and tilted in a geometry previously suggested to occur during intrinsic or Rho-dependent termination [38][39][40] . This active site conformation is consistent with the functional disruption of the nucleotide addition cycle and provides a further structural explanation for the inhibition of catalysis in the absence of bound preQ 1 . ...
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Folding of nascent transcripts can be modulated by the proximal RNA polymerase (RNAP) that carries out their transcription, and vice versa. A pause of RNAP during transcription of a preQ1 riboswitch (que-ePEC) is stabilized by a previously characterized template consensus sequence and the ligand-free conformation of the nascent RNA. Ligand binding to the riboswitch induces RNAP pause release and downstream transcription termination, however, the mechanism by which riboswitch folding modulates pausing is unclear. Here, we report single-particle cryo-electron microscopy reconstructions of que-ePEC in ligand-free and ligand-bound states. In the absence of preQ1, the RNA transcript is in an unexpected hyper-translocated state, preventing downstream nucleotide incorporation. Strikingly, upon ligand binding the riboswitch rotates around its helical axis, expanding the surrounding RNAP exit channel and repositioning the transcript for elongation. Our study reveals the tight coupling by which small nascent RNA structures and their ligands can functionally regulate the macromolecular transcription machinery.
... We assume that the messenger RNA (mRNA) in this model is produced from a nondepleting source. In eukaryotic cells, mRNA is transcribed from a template stand of DNA and a few helicase enzymes have been identified that function to reanneal and rewind the antisense and sense DNA strands upstream of the replication bubble (Park & Roberts, 2006;Wu, 2012). This leaves the promoter region and the gene segment intact for another polymerase to bind and initiate transcription of another mRNA strand. ...
Article
Motivation: Queueing theory can be effective in simulating biochemical reactions taking place in living cells, and the paper paves a step towards development of a comprehensive model of cell metabolism. Such a model could help to accelerate and reduce costs for developing and testing investigational drugs reducing number of laboratory animals needed to evaluate drugs. Results: The paper presents a Krebs cycle model based on queueing theory. The model allows for tracking of metabolites concentration changes in real time. To validate the model, a drug-induced inhibition affecting activity of enzymes involved in Krebs cycle was simulated and compared with available experimental data. Availability: The source code is freely available for download at https://github.com/UTP-WTIiE/KrebsCycleUsingQueueingTheory, implemented in C# supported in Linux or MS Windows. Supplementary information: Supplementary data (tables with kinetic constants, kinetic equations and pseudocode) are available at Bioinformatics online.
... The dissociation of the elongation complex (EC) from DNA requires an active mechanism to destabilize the complex and terminate at a specific position. Intrinsic termination and ρ-dependent termination have evolved as the two dominant pathways in prokaryotic organisms (Park and Roberts, 2006). ...
Thesis
The RNAP (RNA polymerase) is the key enzyme in transcription. RNAP is tightly regulated by factors during transcriptional cycle. Two speed control transcriptional factors (TF) NusA and NusG have the opposite effect on transcriptional pausing. The aim of my work is to use single particle Cryo-EM combine with biochemistry analysis to bring out the regulation of NusA and NusG, and both TFs together on the RNAP. We demonstrate here that RNAP itsself has a constant dynamic movement – non-swiveled to swiveled conformation. NusA stabilized the RNAP to a swiveled conformation which close to the paused state, however NusG enhance the RNAP to an non-swiveled state. NusG-CTD compete with NusA on a same binding site, the Flap-Tip-Helix (FTH) module of RNAP. The biochemistry results showed that these two FT NusA and NusG, compensate the effect of each other and modulate the transcriptional rate in different transcriptional pausing context (class I and class II pausing). However at the termination rho-dependent context, NusA and NusG together could increase the termination efficiency at the terminator I site.
... Mfd is a 132-kDa multi-domain protein that can translocate on DNA, associate with stalled TECs, disassemble TECs to terminate transcription, and recruit the nucleotide-excision repair machinery (Selby and Sancar, 1993;Selby et al., 1991;Witkin, 1966). We focused our nMS screening on variations of the NAS sequences used to assemble a nucleoprotein complex mimicking a stalled TEC that can be processed by Mfd (Komissarova et al., 2003;Park et al., 2002;Park and Roberts, 2006;Vvedenskaya et al., 2014;Zhang et al., 2012). As outlined in Figure S2, we systematically modified the NAS constructs by varying the DNA sequences upstream of the bubble, the DNA sequence complementarity, and the length of the RNA transcript (21-mer or 20-mer for pre-or post-translocated versions, respectively). ...
Article
Recent advances in single-particle cryogenic electron microscopy (cryo-EM) have enabled the structural determination of numerous protein assemblies at high resolution, yielding unprecedented insights into their function. However, despite its extraordinary capabilities, cryo-EM remains time-consuming and resource-intensive. It is therefore beneficial to have a means for rapidly assessing and optimizing the quality of samples prior to lengthy cryo-EM analyses. To do this, we have developed a native mass spectrometry (nMS) platform that provides rapid feedback on sample quality and highly streamlined biochemical screening. Because nMS enables accurate mass analysis of protein complexes, it is well suited to routine evaluation of the composition, integrity, and homogeneity of samples prior to their plunge-freezing on EM grids. We demonstrate the utility of our nMS-based platform for facilitating cryo-EM studies using structural characterizations of exemplar bacterial transcription complexes as well as the replication-transcription assembly from the SARS-CoV-2 virus that is responsible for the COVID-19 pandemic.
... Second, Mfd can rescue backtracked RNAP by promoting forward translocation via ATP hydrolysis (10). Third, Mfd simultaneously interacts with RNAP via the RID and with DNA via the TM, allowing its translocase activity to generate positive torque on the DNA, thereby overwinding the transcription bubble and disrupting the transcription elongation complex (TEC) (12,(14)(15)(16)(17)(18)(19)(20)(21). ...
Article
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Mfd-dependent transcription termination plays an important role in transcription-coupled DNA repair, transcription-replication conflict resolution, and an-timicrobial resistance development. Despite extensive studies, the molecular mechanism of Mfd-dependent transcription termination in bacteria remains unclear, with several long-standing puzzles. How Mfd is activated by stalled RNA polymerase (RNAP) and how activated Mfd translocates along the DNA are unknown. Here, we report the single-particle cryo-electron microscopy structures of T. thermophilus Mfd-RNAP complex with and without ATP␥S. The structures reveal that Mfd undergoes profound conformational changes upon activation, contacts the RNAP ␤1 domain and its clamp, and pries open the RNAP clamp. These structures provide a foundation for future studies aimed at dissecting the precise mechanism of Mfd-dependent transcription termination and pave the way for rational drug design targeting Mfd for the purpose of tackling the antimicrobial resistance crisis.
... Thus, Mfd promotes forward translocation, and it very likely carries out RNA release by this same mechanism: Release is the consequence if the RNAP active center cannot elongate the RNA chain and continue substrate-driven translocation with the full RNA/DNA hybrid, but instead the complex is driven forward by Mfd without RNA synthesis. Rho also was shown to impel forward movement of RNAP as it acts; here the force presumably is imposed on the RNA as the Rho translocase activity pulls it out of the complex (39). Recall that evidence for forward translocation was found specifically for an intrinsic termination site that does not end in a pure homopolymeric sequence, a property also of Rho termination sites. ...
Article
Two strains of good fortune in my career were to stumble upon the Watson–Gilbert laboratory at Harvard when I entered graduate school in 1964, and to study gene regulation in bacteriophage λ when I was there. λ was almost entirely a genetic item a few years before, awaiting biochemical incarnation. Throughout my career I was a relentless consumer of the work of previous and current generations of λ geneticists. Empowered by this background, my laboratory made contributions in two areas. The first was regulation of early gene transcription in λ, the study of which began with the discovery of the Rho transcription termination factor, and the regulatory mechanism of transcription antitermination by the λ N protein, subjects of my thesis work. This was developed into a decades-long program during my career at Cornell, studying the mechanism of transcription termination and antitermination. The second area was the classic problem of prophage induction in response to cellular DNA damage, the study of which illuminated basic cellular processes to survive DNA damage. Expected final online publication date for the Annual Review of Microbiology, Volume 74 is September 8, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Hexameric Rho then uses ATPase activity to translocate 5' to 3' along the RNA. Although the mechanism of Rho-dependent transcription termination is not fully understood, it is thought to occur when Rho reaches the RNA polymerase, e.g. at an RNAP pause site, and forces its movement on template DNA without nucleotide addition, leading to destabilization of the transcription complex and mRNA release [50]. The SNPs identified in rho mapped to different domains of the Rho protein, including an N-terminal insertion domain (NID), primary binding site (PBS), and the C-terminal ATPase domain (Fig 2A). ...
Article
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The intestinal pathogen Clostridioides difficile exhibits heterogeneity in motility and toxin production. This phenotypic heterogeneity is achieved through phase variation by site-specific recombination via the DNA recombinase RecV, which reversibly inverts the “flagellar switch” upstream of the flgB operon. A recV mutation prevents flagellar switch inversion and results in phenotypically locked strains. The orientation of the flagellar switch influences expression of the flgB operon post-transcription initiation, but the specific molecular mechanism is unknown. Here, we report the isolation and characterization of spontaneous suppressor mutants in the non-motile, non-toxigenic recV flg OFF background that regained motility and toxin production. The restored phenotypes corresponded with increased expression of flagellum and toxin genes. The motile suppressor mutants contained single-nucleotide polymorphisms (SNPs) in rho, which encodes the bacterial transcription terminator Rho factor. Analyses using transcriptional reporters indicate that Rho contributes to heterogeneity in flagellar gene expression by preferentially terminating transcription of flg OFF mRNA within the 5’ leader sequence. Additionally, Rho is important for initial colonization of the intestine in a mouse model of infection, which may in part be due to the sporulation and growth defects observed in the rho mutants. Together these data implicate Rho factor as a regulator of gene expression affecting phase variation of important virulence factors of C. difficile.
... This is important not only for early events required to locate RNAP targets on the chromosome, but also for destabilizing TECs halted by DNA damage, pause signals, or various roadblocks 6 . Mfd action on transcription complexes effectively builds up torque to reanneal the upstream edge of the transcription bubble, shorten the bubble, and release the RNA transcript 26,55 . As a first step in shedding light on these events, here we describe the first structure of DNA-bound E. coli Mfd as well as complementary functional analyses. ...
Article
Full-text available
Mfd couples transcription to nucleotide excision repair, and acts on RNA polymerases when elongation is impeded. Depending on impediment severity, this action results in either transcription termination or elongation rescue, which rely on ATP-dependent Mfd translocation on DNA. Due to its role in antibiotic resistance, Mfd is also emerging as a prime target for developing anti-evolution drugs. Here we report the structure of DNA-bound Mfd, which reveals large DNA-induced structural changes that are linked to the active site via ATPase motif VI. These changes relieve autoinhibitory contacts between the N- and C-termini and unmask UvrA recognition determinants. We also demonstrate that translocation relies on a threonine in motif Ic, widely conserved in translocases, and a family-specific histidine near motif IVa, reminiscent of the “arginine clamp” of RNA helicases. Thus, Mfd employs a mode of DNA recognition that at its core is common to ss/ds translocases that act on DNA or RNA.
... Furthermore, comparison with DNAbound UvrD 27 revealed that the template strand would be continuous with a putative HelD-loaded strand, and that conformational changes would be required for HelD to accommodate a DNA strand at D1/D2 in a UvrD-like manner (Fig. 4d). However, as DNA displacement is supported by transcription bubble rewinding 31 , it is unlikely that HelD captures single-stranded DNA at the position revealed in our structure. Our analysis, therefore, indicates that neither DNA binding nor unwinding by HelD is required for RNAP recycling, consistent with lack of helicase activity in isolated HelD 22 . ...
Preprint
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Cellular RNA polymerases can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, or enter dormancy. How RNA polymerase recycling into active states is achieved and balanced with quiescence remains elusive. We structurally analyzed Bacillus subtilis RNA polymerase bound to the NTPase HelD. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNA polymerase secondary channel, dismantling the active site and displacing RNA; a unique helical protrusion inserts into the main channel, prying β and β’ subunits apart and dislodging DNA, aided by the δ subunit. HelD release depends on ATP, and a dimeric structure resembling hibernating RNA polymerase I suggests that HelD can induce dormancy at low energy levels. Our results reveal an ingenious mechanism by which active RNA polymerase pools are adjusted in response to the nutritional state.