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Sivaramesh Wigneshweraraj - One of the best experts on this subject based on the ideXlab platform.
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a bacteriophage dna mimic protein employs a non specific strategy to inhibit the Bacterial rna polymerase
Frontiers in Microbiology, 2021Co-Authors: Zhihao Wang, Sivaramesh Wigneshweraraj, Hongliang Wang, Yawen Wang, Nancy Mulvenna, Maximo Sanzhernandez, Peipei Zhang, Steve Matthews, Bing LiuAbstract:DNA mimicry by proteins is a strategy that employed by some proteins to occupy the binding sites of the DNA-binding proteins and deny further access to these sites by DNA. Such proteins have been found in bacteriophage, eukaryotic virus, prokaryotic, and eukaryotic cells to imitate non-coding functions of DNA. Here, we report another phage protein Gp44 from bacteriophage SPO1 of Bacillus subtilis, employing mimicry as part of unusual strategy to inhibit host RNA polymerase. Consisting of three simple domains, Gp44 contains a DNA binding motif, a flexible DNA mimic domain and a random-coiled domain. Gp44 is able to anchor to host genome and interact Bacterial RNA polymerase via the β and β' subunit, resulting in Bacterial growth inhibition. Our findings represent a non-specific strategy that SPO1 phage uses to target different Bacterial Transcription machinery regardless of the structural variations of RNA polymerases. This feature may have potential applications like generation of genetic engineered phages with Gp44 gene incorporated used in phage therapy to target a range of Bacterial hosts.
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t7 phage factor required for managing rpos inescherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Aline Tabibsalazar, Udi Qimron, Bing Liu, Steve Matthews, Lynn Burchell, Declan Barker, Sivaramesh WigneshwerarajAbstract:T7 development in Escherichia coli requires the inhibition of the housekeeping form of the Bacterial RNA polymerase (RNAP), Eσ70, by two T7 proteins: Gp2 and Gp5.7. Although the biological role of Gp2 is well understood, that of Gp5.7 remains to be fully deciphered. Here, we present results from functional and structural analyses to reveal that Gp5.7 primarily serves to inhibit EσS, the predominant form of the RNAP in the stationary phase of growth, which accumulates in exponentially growing E. coli as a consequence of the buildup of guanosine pentaphosphate [(p)ppGpp] during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development in E. coli cells in the stationary phase of growth. Our finding represents a paradigm for how some lytic phages have evolved distinct mechanisms to inhibit the Bacterial Transcription machinery to facilitate phage development in bacteria in the exponential and stationary phases of growth.
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Conformational heterogeneity and bubble dynamics in single Bacterial Transcription initiation complexes
Nucleic acids research, 2017Co-Authors: Diego Duchi, Konstantin Brodolin, Nicole C. Robb, Kristofer Gryte, Zakia Morichaud, Carol Sheppard, Sivaramesh Wigneshweraraj, Achillefs N. KapanidisAbstract: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 Forster 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.
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a bacteriophage Transcription regulator inhibits Bacterial Transcription initiation by σ factor displacement
Nucleic Acids Research, 2014Co-Authors: B Liu, Carol Sheppard, Konstantin Severinov, A Shadrin, S Matthews, Sivaramesh WigneshwerarajAbstract:Bacteriophages (phages) appropriate essential processes of Bacterial hosts to benefit their own development. The multisubunit Bacterial RNA polymerase (RNAp) enzyme, which catalyses DNA Transcription, is targeted by phage-encoded Transcription regulators that selectively modulate its activity. Here, we describe the structural and mechanistic basis for the inhibition of Bacterial RNAp by the Transcription regulator P7 encoded by Xanthomonas oryzae phage Xp10. We reveal that P7 uses a two-step mechanism to simultaneously interact with the catalytic β and β’ subunits of the Bacterial RNAp and inhibits Transcription initiation by inducing the displacement of the σ70-factor on initial engagement of RNAp with promoter DNA. The new mode of interaction with and inhibition mechanism of Bacterial RNAp by P7 underscore the remarkable variety of mechanisms evolved by phages to interfere with host Transcription.
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modus operandi of the Bacterial rna polymerase containing the σ54 promoter specificity factor
Molecular Microbiology, 2008Co-Authors: Sivaramesh Wigneshweraraj, Daniel Bose, Patricia C Burrows, Nicolas Joly, Jorg Schumacher, Mathieu Rappas, Tillmann Pape, Xiaodong Zhang, Peter G Stockley, Konstantin SeverinovAbstract:Bacterial sigma (sigma) factors confer gene specificity upon the RNA polymerase, the central enzyme that catalyses gene Transcription. The binding of the alternative sigma factor sigma(54) confers upon the RNA polymerase special functional and regulatory properties, making it suited for control of several major adaptive responses. Here, we summarize our current understanding of the interactions the sigma(54) factor makes with the Bacterial Transcription machinery.
Seth A Darst - One of the best experts on this subject based on the ideXlab platform.
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a novel method for the production of in vivo assembled recombinant escherichia coli rna polymerase lacking the α c terminal domain
Protein Science, 2011Co-Authors: Kellyanne F Twist, Bryce E Nickels, Lars F Westblade, Elizabeth A Campbell, Seth A Darst, Seyyed I Husnain, Josef D Franke, Deepti Jain, Mark S ThomasAbstract:The biochemical characterization of the Bacterial Transcription cycle has been greatly facilitated by the production and characterization of targeted RNA polymerase (RNAP) mutants. Traditionally, RNAP preparations containing mutant subunits have been produced by reconstitution of denatured RNAP subunits, a process that is undesirable for biophysical and structural studies. Although schemes that afford the production of in vivo-assembled, recombinant RNAP containing amino acid substitutions, insertions, or deletions in either the monomeric β or β′ subunits have been developed, there is no such system for the production of in vivo-assembled, recombinant RNAP with mutations in the homodimeric α-subunits. Here, we demonstrate a strategy to generate in vivo-assembled, recombinant RNAP preparations free of the α C-terminal domain. Furthermore, we describe a modification of this approach that would permit the purification of in vivo-assembled, recombinant RNAP containing any α-subunit variant, including those variants that are lethal. Finally, we propose that these related approaches can be extended to generate in vivo-assembled, recombinant variants of other protein complexes containing homomultimers for biochemical, biophysical, and structural analyses.
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structural basis for the Bacterial Transcription repair coupling factor rna polymerase interaction
Nucleic Acids Research, 2010Co-Authors: Lars F Westblade, Bryce E Nickels, Elizabeth A Campbell, Chirangini Pukhrambam, Julio C Padovan, Valerie Lamour, Seth A DarstAbstract:The Transcription-repair coupling factor (TRCF, the product of the mfd gene) is a widely conserved Bacterial protein that mediates Transcription-coupled DNA repair. TRCF uses its ATP-dependent DNA translocase activity to remove Transcription complexes stalled at sites of DNA damage, and stimulates repair by recruiting components of the nucleotide excision repair pathway to the site. A protein/protein interaction between TRCF and the β-subunit of RNA polymerase (RNAP) is essential for TRCF function. CarD (also called CdnL), an essential regulator of rRNA Transcription in Mycobacterium tuberculosis, shares a homologous RNAP interacting domain with TRCF and also interacts with the RNAP β-subunit. We determined the 2.9-A resolution X-ray crystal structure of the RNAP interacting domain of TRCF complexed with the RNAP-β1 domain, which harbors the TRCF interaction determinants. The structure reveals details of the TRCF/RNAP protein/protein interface, providing a basis for the design and interpretation of experiments probing TRCF, and by homology CarD, function and interactions with the RNAP.
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structural basis for Bacterial Transcription coupled dna repair
Cell, 2006Co-Authors: Alexandra M Deaconescu, Bryce E Nickels, Anna L Chambers, Abigail J Smith, Ann Hochschild, Nigel J Savery, Seth A DarstAbstract:Coupling of Transcription and DNA repair in bacteria is mediated by Transcription-repair coupling factor (TRCF, the product of the mfd gene), which removes Transcription elongation complexes stalled at DNA lesions and recruits the nucleotide excision repair machinery to the site. Here we describe the 3.2 A-resolution X-ray crystal structure of Escherichia coli TRCF. The structure consists of a compact arrangement of eight domains, including a translocation module similar to the SF2 ATPase RecG, and a region of structural similarity to UvrB. Biochemical and genetic experiments establish that another domain with structural similarity to the Tudor-like domain of the Transcription elongation factor NusG plays a critical role in TRCF/RNA polymerase interactions. Comparison with the translocation module of RecG as well as other structural features indicate that TRCF function involves large-scale conformational changes. These data, along with a structural model for the interaction of TRCF with the Transcription elongation complex, provide mechanistic insights into TRCF function.
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structure of the Bacterial rna polymerase promoter specificity σ subunit
Molecular Cell, 2002Co-Authors: Elizabeth A Campbell, Oriana Muzzin, Mark Chlenov, J L Sun, Anders C Olson, Oren Weinman, Michelle Tresterzedlitz, Seth A DarstAbstract:Abstract The σ subunit is the key regulator of Bacterial Transcription. Proteolysis of Thermus aquaticus σ A , which occurred in situ during crystallization, reveals three domains, σ 2 , σ 3 , and σ 4 , connected by flexible linkers. Crystal structures of each domain were determined, as well as of σ 4 complexed with −35 element DNA. Exposed surfaces of each domain are important for RNA polymerase binding. Universally conserved residues important for −10 element recognition and melting lie on one face of σ 2 , while residues important for extended −10 recognition lie on σ 3 . Genetic studies correctly predicted that a helix-turn-helix motif in σ 4 recognizes the −35 element but not the details of the protein-DNA interactions. Positive control mutants in σ 4 cluster in two regions, positioned to interact with activators bound just upstream or downstream of the −35 element.
Cherry Gao - One of the best experts on this subject based on the ideXlab platform.
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Single-cell Bacterial Transcription measurements reveal the importance of dimethylsulfoniopropionate (DMSP) hotspots in ocean sulfur cycling.
Nature communications, 2020Co-Authors: Cherry Gao, Georg Pohnert, Vicente Fernandez, Kang Soo Lee, Simona Fenizia, Justin R. Seymour, Jean-baptiste Raina, Roman StockerAbstract:Dimethylsulfoniopropionate (DMSP) is a pivotal compound in marine biogeochemical cycles and a key chemical currency in microbial interactions. Marine bacteria transform DMSP via two competing pathways with considerably different biogeochemical implications: demethylation channels sulfur into the microbial food web, whereas cleavage releases sulfur into the atmosphere. Here, we present single-cell measurements of the expression of these two pathways using engineered fluorescent reporter strains of Ruegeria pomeroyi DSS-3, and find that external DMSP concentration dictates the relative expression of the two pathways. DMSP induces an upregulation of both pathways, but only at high concentrations (>1 μM for demethylation; >35 nM for cleavage), characteristic of microscale hotspots such as the vicinity of phytoplankton cells. Co-incubations between DMSP-producing microalgae and bacteria revealed an increase in cleavage pathway expression close to the microalgae's surface. These results indicate that Bacterial utilization of microscale DMSP hotspots is an important determinant of the fate of sulfur in the ocean.
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single cell Bacterial Transcription measurements reveal the importance of dimethylsulfoniopropionate dmsp hotspots in ocean sulfur cycling
Nature Communications, 2020Co-Authors: Georg Pohnert, Cherry Gao, Vicente Fernandez, Kang Soo Lee, Simona Fenizia, Justin R. Seymour, Jean-baptiste Raina, Roman StockerAbstract:Dimethylsulfoniopropionate (DMSP) is a pivotal compound in marine biogeochemical cycles and a key chemical currency in microbial interactions. Marine bacteria transform DMSP via two competing pathways with considerably different biogeochemical implications: demethylation channels sulfur into the microbial food web, whereas cleavage releases sulfur into the atmosphere. Here, we present single-cell measurements of the expression of these two pathways using engineered fluorescent reporter strains of Ruegeria pomeroyi DSS-3, and find that external DMSP concentration dictates the relative expression of the two pathways. DMSP induces an upregulation of both pathways, but only at high concentrations (>1 μM for demethylation; >35 nM for cleavage), characteristic of microscale hotspots such as the vicinity of phytoplankton cells. Co-incubations between DMSP-producing microalgae and bacteria revealed an increase in cleavage pathway expression close to the microalgae’s surface. These results indicate that Bacterial utilization of microscale DMSP hotspots is an important determinant of the fate of sulfur in the ocean. DMSP is a ubiquitous organosulfur compound in the ocean that, once degraded by bacteria, plays key roles in global biogeochemical cycles and climate regulation. Here, the authors use single-cell measurements of Transcription to investigate the intricate dynamics of Bacterial DMSP degradation.
Roman Stocker - One of the best experts on this subject based on the ideXlab platform.
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Single-cell Bacterial Transcription measurements reveal the importance of dimethylsulfoniopropionate (DMSP) hotspots in ocean sulfur cycling.
Nature communications, 2020Co-Authors: Cherry Gao, Georg Pohnert, Vicente Fernandez, Kang Soo Lee, Simona Fenizia, Justin R. Seymour, Jean-baptiste Raina, Roman StockerAbstract:Dimethylsulfoniopropionate (DMSP) is a pivotal compound in marine biogeochemical cycles and a key chemical currency in microbial interactions. Marine bacteria transform DMSP via two competing pathways with considerably different biogeochemical implications: demethylation channels sulfur into the microbial food web, whereas cleavage releases sulfur into the atmosphere. Here, we present single-cell measurements of the expression of these two pathways using engineered fluorescent reporter strains of Ruegeria pomeroyi DSS-3, and find that external DMSP concentration dictates the relative expression of the two pathways. DMSP induces an upregulation of both pathways, but only at high concentrations (>1 μM for demethylation; >35 nM for cleavage), characteristic of microscale hotspots such as the vicinity of phytoplankton cells. Co-incubations between DMSP-producing microalgae and bacteria revealed an increase in cleavage pathway expression close to the microalgae's surface. These results indicate that Bacterial utilization of microscale DMSP hotspots is an important determinant of the fate of sulfur in the ocean.
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single cell Bacterial Transcription measurements reveal the importance of dimethylsulfoniopropionate dmsp hotspots in ocean sulfur cycling
Nature Communications, 2020Co-Authors: Georg Pohnert, Cherry Gao, Vicente Fernandez, Kang Soo Lee, Simona Fenizia, Justin R. Seymour, Jean-baptiste Raina, Roman StockerAbstract:Dimethylsulfoniopropionate (DMSP) is a pivotal compound in marine biogeochemical cycles and a key chemical currency in microbial interactions. Marine bacteria transform DMSP via two competing pathways with considerably different biogeochemical implications: demethylation channels sulfur into the microbial food web, whereas cleavage releases sulfur into the atmosphere. Here, we present single-cell measurements of the expression of these two pathways using engineered fluorescent reporter strains of Ruegeria pomeroyi DSS-3, and find that external DMSP concentration dictates the relative expression of the two pathways. DMSP induces an upregulation of both pathways, but only at high concentrations (>1 μM for demethylation; >35 nM for cleavage), characteristic of microscale hotspots such as the vicinity of phytoplankton cells. Co-incubations between DMSP-producing microalgae and bacteria revealed an increase in cleavage pathway expression close to the microalgae’s surface. These results indicate that Bacterial utilization of microscale DMSP hotspots is an important determinant of the fate of sulfur in the ocean. DMSP is a ubiquitous organosulfur compound in the ocean that, once degraded by bacteria, plays key roles in global biogeochemical cycles and climate regulation. Here, the authors use single-cell measurements of Transcription to investigate the intricate dynamics of Bacterial DMSP degradation.
Marc Boudvillain - One of the best experts on this subject based on the ideXlab platform.
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A simple fluorescence microplate assay to monitor RNA-DNA hybrid unwinding by the Bacterial Transcription termination factor Rho
2021Co-Authors: Isabelle Simon, Marc BoudvillainAbstract:Transcription termination factor Rho contributes to shape the transcriptomes of many bacteria and is essential in a large subset of them. Although the Transcription termination function of Rho is not always easy to reconstitute and to study in vitro, assays based on the ATPdependent RNA-DNA hybrid unwinding activity of the factor can prove useful to dissect Rho mechanisms or to seek new antibiotics targeting Rho. However, current in vitro assays of Rho helicase activity are time-consuming, as they usually require radiolabeling of the hybrid substrates and analysis of reaction products by gel electrophoresis. Here, we describe a fluorescence-based microplate assay that informs on Rho helicase activity in a matter of minutes and allows the multiplexed analysis of conditions required for primary biochemical characterization or for drug screening.
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a multivariate prediction model for rho dependent termination of Transcription
Nucleic Acids Research, 2018Co-Authors: Cedric Nadiras, Annie Schwartz, Nara Figueroabossi, Eric Eveno, Marc BoudvillainAbstract:: Bacterial Transcription termination proceeds via two main mechanisms triggered either by simple, well-conserved (intrinsic) nucleic acid motifs or by the motor protein Rho. Although Bacterial genomes can harbor hundreds of termination signals of either type, only intrinsic terminators are reliably predicted. Computational tools to detect the more complex and diversiform Rho-dependent terminators are lacking. To tackle this issue, we devised a prediction method based on Orthogonal Projections to Latent Structures Discriminant Analysis [OPLS-DA] of a large set of in vitro termination data. Using previously uncharacterized genomic sequences for biochemical evaluation and OPLS-DA, we identified new Rho-dependent signals and quantitative sequence descriptors with significant predictive value. Most relevant descriptors specify features of transcript C>G skewness, secondary structure, and richness in regularly-spaced 5'CC/UC dinucleotides that are consistent with known principles for Rho-RNA interaction. Descriptors collectively warrant OPLS-DA predictions of Rho-dependent termination with a ∼85% success rate. Scanning of the Escherichia coli genome with the OPLS-DA model identifies significantly more termination-competent regions than anticipated from transcriptomics and predicts that regions intrinsically refractory to Rho are primarily located in open reading frames. Altogether, this work delineates features important for Rho activity and describes the first method able to predict Rho-dependent terminators in Bacterial genomes.
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ATP-dependent motor activity of the Transcription termination factor Rho from Mycobacterium tuberculosis
Nucleic Acids Research, 2015Co-Authors: François D'heygère, Annie Schwartz, Franck Coste, Bertrand Castaing, Marc BoudvillainAbstract:The Bacterial Transcription termination factor Rho—a ring-shaped molecular motor displaying directional, ATP-dependent RNA helicase/translocase activity—is an interesting therapeutic target. Recently, Rho from Mycobacterium tuberculosis (MtbRho) has been proposed to operate by a mechanism uncoupled from molecular motor action, suggesting that the manner used by Rho to dissociate Transcriptional complexes is not conserved throughout the Bacterial kingdom. Here, however, we demonstrate that MtbRho is a bona fide molecular motor and directional helicase which requires a catalytic site competent for ATP hydrolysis to disrupt RNA duplexes or Transcription elongation complexes. Moreover, we show that idiosyncratic features of the MtbRho enzyme are conferred by a large, hydrophilic insertion in its N-terminal ‘RNA binding’ domain and by a non-canonical R-loop residue in its C-terminal ‘motor’ domain. We also show that the ‘motor’ domain of MtbRho has a low apparent affinity for the Rho inhibitor bicyclomycin, thereby contributing to explain why M. tuberculosis is resistant to this drug. Overall, our findings support that, in spite of adjustments of the Rho motor to specific traits of its hosting bacterium, the basic principles of Rho action are conserved across species and could thus constitute pertinent screening criteria in high-throughput searches of new Rho inhibitors.
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phyletic distribution and conservation of the Bacterial Transcription termination factor rho
Microbiology, 2013Co-Authors: Marc Boudvillain, Francois Dheygere, Makhlouf RabhiAbstract:Transcription termination factor Rho is a ring-shaped, ATP-dependent molecular motor that targets hundreds of Transcription units in Escherichia coli. Interest in Rho was renewed recently on the realization that this essential factor is involved in multiple interactions and cellular processes that protect the E. coli genome and regulate its expression on a global scale. Yet it is currently unknown if (and how) Rho-dependent mechanisms are conserved throughout the Bacterial kingdom. Here, we mined public databases to assess the distribution, expression and structural conservation of Rho across Bacterial phyla. We found that rho is present in more than 90 % of sequenced Bacterial genomes, although Cyanobacteria, Mollicutes and a fraction of Firmicutes are totally devoid of rho. Genomes lacking rho tend to be small and AT-rich and often belong to species with parasitic/symbiotic lifestyles (such as Mollicutes). By contrast, large GC-rich genomes, such as those of Actinobacteria, often contain rho duplicates and/or encode Rho proteins that bear insertion domains of unknown function(s). Notwithstanding, most Rho sequences across taxa contain canonical RNA-binding and ATP hydrolysis signature motifs, a feature suggestive of largely conserved mechanism(s) of action. Mutations that impair binding of bicyclomycin are present in ~5 % of rho sequences, implying that species from diverse ecosystems have developed resistance against this natural antibiotic. Altogether, these findings assert that Rho function is widespread among bacteria and suggest that it plays a particularly relevant role in the expression of complex genomes and/or Bacterial adaptation to changing environments.
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a stepwise 2 hydroxyl activation mechanism for the Bacterial Transcription termination factor rho helicase
Nature Structural & Molecular Biology, 2009Co-Authors: Annie Schwartz, Emmanuel Margeat, Makhlouf Rabhi, Frederique Jacquinot, Rachid A Rahmouni, Marc BoudvillainAbstract:The Bacterial Rho factor is a ring-shaped ATP-dependent helicase that tracks along RNA transcripts and disrupts RNA-DNA duplexes and Transcription complexes in its path. Using combinatorial nucleotide analog interference mapping (NAIM), we explore the topology and dynamics of functional Rho-RNA complexes and reveal the RNA-dependent stepping mechanism of Rho helicase. Periodic Gaussian distributions of NAIM signals show that Rho forms uneven productive interactions with the track nucleotides and disrupts RNA-DNA duplexes in a succession of large ( approximately 7-nucleotide-long) discrete steps triggered by 2'-hydroxyl activation events. This periodic 2'-OH-dependent activation does not depend on the RNA-DNA pairing energy but is finely tuned by sequence-dependent interactions with the RNA track. These features explain the strict RNA specificity and contextual efficiency of the enzyme and provide a new paradigm for conditional tracking by a helicase ring.