Thermodynamic and kinetic modeling of transcriptional pausing

Tadigotla, Vasisht; Maoiléidigh, Dáibhid Ó; Sengupta, Anirvan M.; Epshtein, Vitaly; Ebright, Richard H.; Nudler, Evgeny; Ruckenstein, Andrei E.

doi:10.1073/pnas.0600508103

Cited by 106 publications

(192 citation statements)

References 46 publications

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“…One mechanism by which the template composition can produce pause attenuation-reducing the rate of pause entry and the pause duration-is if the secondary structure of the nascent RNA upstream of the polymerase can act as an energy barrier to backtracking (17,30), because GC-rich RNA sequences form more stable secondary structures than AU-rich ones. According to this hypothesis, backtracking, and therefore pause duration and density, should be reduced on templates with larger GC content.…”

Section: Resultsmentioning

confidence: 99%

Nascent RNA structure modulates the transcriptional dynamics of RNA polymerases

Zamft

Bintu

Ishibashi

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

100

127

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RNA polymerase pausing represents an important mechanism of transcriptional regulation. In this study, we use a single-molecule transcription assay to investigate the effect of template base-pair composition on pausing by RNA polymerase II and the evolutionarily distinct mitochondrial polymerase Rpo41. For both enzymes, pauses are shorter and less frequent on GC-rich templates. Significantly, incubation with RNase abolishes the template dependence of pausing. A kinetic model, wherein the secondary structure of the nascent RNA poses an energetic barrier to pausing by impeding backtracking along the template, quantitatively predicts the pause densities and durations observed. The energy barriers extracted from the data correlate well with RNA folding energies obtained from cotranscriptional folding simulations. These results reveal that RNA secondary structures provide a cis-acting mechanism by which sequence modulates transcriptional elongation.enzyme kinetics | Pol II | transcriptional pausing | yeast T ranscription represents the first point of control of gene expression. Its regulation determines the RNA levels in the cell and, ultimately, such varied processes as cell-cycle coordination, metabolism, growth, and death. Regulation of RNA throughput occurs at all stages of the transcription process, i.e., initiation, elongation, and termination (1). Regulation at initiation involves complex machinery that assembles at the promoter and endows the cell with the capacity to make a binary decision in response to internal or external signals. Similarly, transcriptional termination requires a binary decision at specific locations in the sequence. In contrast, regulation of the elongation phase is spatially distributed throughout the transcribed gene and involves the modulation of the dynamics of RNA synthesis by cis-and trans-acting factors.One of the most prominent aspects of the dynamics of RNA polymerases is their tendency to pause. Pausing is an intrinsic property of most RNA polymerases, and its regulation constitutes one of the central mechanisms of control of gene expression. Sequence-specific pausing allows for the recruitment of trans-acting elements that are implicated in promoter escape (2), alternative splicing (3), factor-dependent termination (4), proofreading (5), and further transcriptional regulation (6-9). Pauses also play a role in factor-independent termination, which is mediated by secondary structures of the nascent RNA near the termination site (10). In contrast to the punctate nature of pausing during initiation and termination, pauses during elongation can, in general, occur anywhere along the transcribed gene.The finding that pause durations of RNAP II follow a t −3∕2 distribution (to first order) implies that pausing occurs through a diffusive mechanism (5). This observation, along with additional evidence of backward movement of RNA polymerases on their template (11,12), has led to the backtracking model of transcriptional pausing. In this model, pauses are initiated by retrograde movement ...

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Section: Resultsmentioning

confidence: 99%

Nascent RNA structure modulates the transcriptional dynamics of RNA polymerases

Zamft

Bintu

Ishibashi

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

100

127

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“…Surprisingly, in spite of the close similarities between the template-dictated and motor-driven polymerization of macromolecules in transcription and translation, no attempt has been made in the past to incorporate interactions of RNAPs in the theoretical description of transcription. Instead, to our knowledge, all the models of transcription reported so far [27,28,29,30,31,32,33,34,35,36,37,38] capture only the stochatic mechano-chemistry of the individual RNAP motors. Cooperation and collisions between RNAP motors is known to have non-trivial effects on the rate of transcription [39,40,41,42].…”

Section: Introductionmentioning

confidence: 99%

Interacting RNA polymerase motors on a DNA track: Effects of traffic congestion and intrinsic noise on RNA synthesis

Tripathi

Chowdhury

2008

Phys. Rev. E

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RNA polymerase (RNAP) is an enzyme that synthesizes a messenger RNA (mRNA) strand which is complementary to a single-stranded DNA template. From the perspective of physicists, an RNAP is a molecular motor that utilizes chemical energy input to move along the track formed by a DNA. In many circumstances, which are described in this paper, a large number of RNAPs move simultaneously along the same track; we refer to such collective movements of the RNAPs as RNAP traffic. Here we develop a theoretical model for RNAP traffic by incorporating the steric interactions between RNAPs as well as the mechano-chemical cycle of individual RNAPs during the elongation of the mRNA. By a combination of analytical and numerical techniques, we calculate the rates of mRNA synthesis and the average density profile of the RNAPs on the DNA track. We also introduce, and compute, two new measures of fluctuations in the synthesis of RNA. Analyzing these fluctuations, we show how the level of intrinsic noise in mRNA synthesis depends on the concentrations of the RNAPs as well as on those of some of the reactants and the products of the enzymatic reactions catalyzed by RNAP. We suggest appropriate experimental systems and techniques for testing our theoretical predictions.

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“…Thus sequences that weaken upstream bubble collapse may lead to longer lifetimes, whereas sequences that favor melting downstream of the bubble may lead to shorter lifetimes. In predicting such effects, one will have to include not only dinucleotide step thermodynamics but also the energetic sequencedependence of the edges of the bubbles, perhaps approximated by thermodynamic studies of dangling ends (23,24,26,27). In addition, these duplexes, whether at the upstream or downstream edges of the bubble or in the RNA-DNA hybrid, are not free in solution but may interact with the protein in ways not accounted for by solutionphase model duplexes.…”

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confidence: 99%

Dissociation of halted T7 RNA polymerase elongation complexes proceeds via a forward-translocation mechanism

Zhou¹,

Navaroli²,

Enuameh

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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hypertranslocation ͉ hybrid ͉ stability ͉ topological lock T ranscription lies at the heart of cellular gene expression. Unlike replication, transcription is not distributive; that is, any dissociation of an elongation complex is a terminal event. Consequently, elongation complexes must be highly stable while transcribing at up to several hundred bases per second. Recent studies demonstrate, however, that elongation is not a uniform process, with reasonably long-lived, sequence-dependent pauses occurring stochastically (1-4). Indeed, the elongation phase is well known to be subject to regulation through attenuation: sequence-dependent signals leading to pause, arrest, slippage, or termination (5). Elongating RNA polymerases must hold the DNA template and the RNA product tightly enough to be highly stable through nonterminating pauses, yet they must be able to dissociate the complex in response to specific sequences in the DNA. Understanding this interplay requires understanding mechanisms of dissociation.In classic rho-independent or ''intrinsic'' termination, dissociation is thought to occur as the polymerase slows in response to sequence, and structure begins to form concurrently in the nascent transcript (6-8). A run of encoded U's has been proposed both to slow transcription and to weaken the RNA-DNA hybrid. Recently, it has been proposed that, rather than direct dissociation of the nascent transcript from a complex halted at the primary termination site (with or without allosteric assistance), dissociation occurs primarily from forward-translocated states (9-11). In forwardtranslocated (hypertranslocated) states, the hybrid is shortened, weakening the binding of the RNA to the complex. Additionally, shortening of the hybrid may also serve to remove a topological locking of the RNA onto the template strand. As illustrated in Fig. 1, dissociation of the RNA transcript would be substantially faster from forward translocated states than from the initially paused state. Although forward-translocated states are expected to be energetically disfavored relative to the initial on-pathway state and so would be expected to exist at lower populations, the difference in the kinetics of dissociation could compensate such that the dominant dissociative pathway proceeds via forward translocated states.The model shown in Fig. 1 predicts certain behaviors. In general, forward translocation requires melting of the DNA duplex downstream and dissociation of the most upstream base(s) in the hybrid. These costs are partially offset by the energy gained by collapse of a base pair in the DNA at the upstream edge of the bubble. Thus, one would expect that altering the balance between these effects could shift the distribution toward more forward-translocated states and therefore toward or away from dissociation. As demonstrated previously, a direct crosslink between the DNA strands downstream of the halt site should prevent forward translocation and is in fact seen to reduce termination from an intrinsic terminator (9).We propose t...

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Thermodynamic and kinetic modeling of transcriptional pausing

Cited by 106 publications

References 46 publications

Nascent RNA structure modulates the transcriptional dynamics of RNA polymerases

Nascent RNA structure modulates the transcriptional dynamics of RNA polymerases

Interacting RNA polymerase motors on a DNA track: Effects of traffic congestion and intrinsic noise on RNA synthesis

Dissociation of halted T7 RNA polymerase elongation complexes proceeds via a forward-translocation mechanism

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