Accounting for RNA polymerase heterogeneity reveals state switching and two distinct long-lived backtrack states escaping through cleavage

Janissen, Richard; Eslami-Mossallam, Behrouz; Artsimovitch, Irina; Depken, Martin; Nh, Dekker

doi:10.1101/762468

Cited by 3 publications

(2 citation statements)

References 97 publications

(468 reference statements)

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“…Furthermore, we measured up to 300 DNA tethers simultaneously with instrumental drift compensation at a frequency of 50 Hz, which provides large data sets suited for statistically robust analysis Janissen et al, 2018Janissen et al, , 2020.…”

Section: Magnetic Tweezers Resolve Single Dna Extrusion Steps Of Indimentioning

confidence: 99%

Condensin extrudes DNA loops in steps up to hundreds of base pairs that are generated by ATP binding events

Ryu

Rah

Janissen

et al. 2020

Preprint

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The condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ATP-dependent motor mechanism remains unclear but likely involves steps associated with large conformational changes within the ~50 nm protein complex. Here, using magnetic tweezers, we resolve single steps in loop extrusion by individual yeast condensins. Step sizes range between 20-45 nm at forces of 1.0-0.2 pN, respectively, comparable to the complex size. The large steps show that condensin reels in DNA in very sizeable amounts, up to ~600 bp per extrusion step, consistent with the non-stretched DNA polymer at these low forces. Using ATP-binding-impaired and ATP-hydrolysis-deficient mutants, we find that ATP binding is the primary step-generating stage underlying DNA loop extrusion. We discuss the findings in terms of a scrunching model where a stepwise DNA loop extrusion is generated by an ATP-binding-induced engagement of the hinge and the globular domain of the SMC complex.

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Section: Magnetic Tweezers Resolve Single Dna Extrusion Steps Of Indimentioning

confidence: 99%

Condensin extrudes DNA loops in steps up to hundreds of base pairs that are generated by ATP binding events

Ryu

Rah

Janissen

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Using magnetic tweezers, pausing events of bacterial RNA polymerase could be studied with a high temporal bandwidth (2.5h with 40ms resolution, i.e. 5 orders in time), which revealed considerable heterogeneity in transcription velocity and pause dynamics [95]. While the causative internal rearrangements of the polymerase remain elusive in the magnetic tweezers assay, they could be resolved in real-time smFRET recordings [92], albeit at a much-reduced temporal bandwidth.…”

Section: Biological Use Casesmentioning

confidence: 99%

Can DyeCycling break the photobleaching limit in single-molecule FRET?

Vermeer

Schmid

2022

Preprint

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Biomolecular systems, such as proteins, crucially rely on dynamic processes at the nanoscale. Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular driving forces that control biomolecular systems. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to observe in real-time how a single biomolecule proceeds through its functional cycle involving a sequence of distinct structural states. Currently, this technique is fundamentally limited by irreversible photobleaching, causing the untimely end of the experiment and thus, a prohibitively narrow temporal bandwidth of ≤ 3 orders of magnitude. Here, we introduce ″DyeCycling″, a measurement scheme with which we aim to break the photobleaching limit in single-molecule FRET. We introduce the concept of spontaneous dye replacement by simulations, and as an experimental proof-of-concept, we demonstrate the intermittent observation of a single biomolecule for one hour with a time resolution of milliseconds. Theoretically, DyeCycling can provide >100-fold more information per single molecule than conventional smFRET. We discuss the experimental implementation of DyeCycling, its current and fundamental limitations, and specific biological use cases. Given its general simplicity and versatility, DyeCycling has the potential to revolutionize the field of time-resolved smFRET, where it may serve to unravel a wealth of biomolecular dynamics by bridging from milliseconds to the hour range.

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Basic mechanisms and kinetics of pause-interspersed transcript elongation

Qian

Dunlap

Finzi

2020

Nucleic Acids Research

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RNA polymerase pausing during elongation is an important mechanism in the regulation of gene expression. Pausing along DNA templates is thought to be induced by distinct signals encoded in the nucleic acid sequence and halt elongation complexes to allow time for necessary co-transcriptional events. Pausing signals have been classified as those producing short-lived elemental, long-lived backtracked, or hairpin-stabilized pauses. In recent years, structural microbiology and single-molecule studies have significantly advanced our understanding of the paused states, but the dynamics of these states are still uncertain, although several models have been proposed to explain the experimentally observed pausing behaviors. This review summarizes present knowledge about the paused states, discusses key discrepancies among the kinetic models and their basic assumptions, and highlights the importance and challenges in constructing theoretical models that may further our biochemical understanding of transcriptional pausing.

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Accounting for RNA polymerase heterogeneity reveals state switching and two distinct long-lived backtrack states escaping through cleavage

Cited by 3 publications

References 97 publications

Condensin extrudes DNA loops in steps up to hundreds of base pairs that are generated by ATP binding events

Condensin extrudes DNA loops in steps up to hundreds of base pairs that are generated by ATP binding events

Can DyeCycling break the photobleaching limit in single-molecule FRET?

Basic mechanisms and kinetics of pause-interspersed transcript elongation

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