Recent Advances in Understanding σ70-Dependent Transcription Initiation Mechanisms

Mazumder, Abhishek; Kapanidis, Achillefs N.

doi:10.1016/j.jmb.2019.04.046

Cited by 64 publications

(73 citation statements)

References 79 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…for example persistence length (l p ) and salt-dependent changes in l p ) of DNA accurately. Predictions using the model 36 for bubble formation in promoter DNA during bacterial transcription have found experimental support,39,40 thus validating the model. In the SOP model, the center of a bead representing the protein residue is at the C α atom, and the bead representing the nucleotide is at its center of mass.…”

mentioning

confidence: 63%

Asymmetry in Histone Rotation in Forced Unwrapping and Force Quench Rewrapping in a Nucleosome

Reddy¹,

Thirumalai

2020

Preprint

View full text Add to dashboard Cite

Nucleosomes, the building blocks of chromosomes, are also transcription regulators. Single molecule pulling experiments have shown that nucleosomes unwrap in two major stages, releasing nearly equal length of DNA in each stage. The first stage, attributed to the rupture of the outer turn is reversible, occurs at low forces (≈ (3 - 5) pNs) whereas in the second stage the inner turn ruptures irreversibly at high forces (between ≈ (9 - 15) or higher) pNs. We show that Brownian dynamics simulations using the Self-Organized Polymer model of the nucleosome capture the experimental findings, thus permitting us to discern the molecular details of the structural changes not only in DNA but also in the Histone Protein Core (HPC). Upon unwrapping of the outer turn, which is independent of the pulling direction, there is a transition from 1.6 turns to 1.0 turn DNA wound around the HPC. In contrast, the rupture of the inner turn, leading to less than 0.5 turn DNA around the HPC, depends on the pulling direction, and is controlled by energetic and kinetic barriers. The latter arises because the mechanical force has to produce sufficient torque to rotate (in an almost directed manner) the HPC by 180°. In contrast, during the rewrapping process, HPC rotation is stochastic, with the quenched force fQ playing no role. Interestingly, if fQ = 0 the HPC rotation is not required for rewrapping because the DNA ends are unconstrained. The assembly of the outer wrap upon force quench, as assessed by the decrease in the end-to-end distance (Ree) of the DNA, nearly coincides with the increase in Ree as force is increased, confirming the reversible nature of the 1.6 turns to 1.0 turn transition. The asymmetry in HPC rotation during unwrapping and rewrapping accounts for the observed hysteresis in the stretch-release cycles in single molecule pulling experiments. Experiments that could validate the prediction that HPC rotation, which gives rise to the kinetic barrier in the unwrapping process, are proposed.

show abstract

mentioning

confidence: 63%

Asymmetry in Histone Rotation in Forced Unwrapping and Force Quench Rewrapping in a Nucleosome

Reddy¹,

Thirumalai

2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Initial transcription proceeds until synthesis of an RNA product of a threshold length of ~10 nt (Mazumder and Kapanidis, 2019;Winkelman et al, 2019). Upon synthesis of a threshold-length RNA product, RNAP breaks the sequence-specific protein-DNA interactions that anchor it on promoter DNA, escapes the promoter, and synthesizes the rest of the RNA product as a transcription elongation complex (TEC; Figure 1B).…”

Section: Introductionmentioning

confidence: 99%

XACT-seq comprehensively defines the promoter-position and promoter-sequence determinants for initial-transcription pausing

Winkelman

Pukhrambam

Vvedenskaya

et al. 2019

Preprint

View full text Add to dashboard Cite

Pausing by RNA polymerase (RNAP) during transcription elongation, in which a translocating RNAP uses a "stepping" mechanism, has been studied extensively, but pausing by RNAP during initial transcription, in which a promoter-anchored RNAP uses a "scrunching" mechanism, has not. We report a method that directly defines RNAP-active-center position relative to DNA in vivo with single-nucleotide resolution (XACT-seq;crosslink-between-active-center-and-template sequencing). We apply this method to detect and quantify pausing in initial transcription at 4 11 (~4,000,000) promoter sequences in vivo, in Escherichia coli. The results show initial-transcription pausing can occur in each nucleotide addition during initial transcription, particularly the first 4-5 nucleotide additions. The results further show initial-transcription pausing occurs at sequences that resemble the consensus sequence element for transcription-elongation pausing. Our findings define the positional and sequence determinants for initial-transcription pausing and establish initial-transcription pausing is hard-coded by sequence elements similar to those for transcription-elongation pausing.

show abstract

“…RNA synthesis by all DNA-dependent RNA polymerases (RNAP) is a tightly regulated dynamic process involving large-scale conformational changes in both the enzyme and DNA. Mechanistic investigations of the formation of the transcriptionally-competent open complex, Rpo, by bacterial RNAP have defined a multi-step pathway where a series of intermediates appear and disappear on the subsecond to seconds time scale 32, 33 . While “kinetically-significant” intermediates in RPo formation were identified decades ago 34 , their transient nature has prevented atomic resolution structural characterization.…”

Section: Figurementioning

confidence: 99%

Time-resolved cryoEM using Spotiton

Dandey

Wei

Bobe

et al. 2020

Preprint

View full text Add to dashboard Cite

28We present an approach for preparing cryoEM grids to study short-lived molecular states. 29Using piezo electric dispensing, two independent streams of ~50 pL sample drops are 30 deposited within 10 ms of each other onto a nanowire EM grid surface, and the mixing 31 reaction stops when the grid is vitrified in liquid ethane, on the order of ~100 ms later. We 32 demonstrate the utility of this approach for four biological systems where short-lived 33states are of high interest. 34 38 Chen, J. et al. E. coli TraR allosterically regulates transcription initiation by altering RNA 541 polymerase conformation. eLife 8, doi:10.7554/eLife.49375 (2019). 542 39 Posson, D. J., Rusinova, R., Andersen, O. S. & Nimigean, C. M. Calcium ions open a 543 selectivity filter gate during activation of the MthK potassium channel. Nat Commun 6, 544 8342,

show abstract

Recent Advances in Understanding σ70-Dependent Transcription Initiation Mechanisms

Cited by 64 publications

References 79 publications

Asymmetry in Histone Rotation in Forced Unwrapping and Force Quench Rewrapping in a Nucleosome

Asymmetry in Histone Rotation in Forced Unwrapping and Force Quench Rewrapping in a Nucleosome

XACT-seq comprehensively defines the promoter-position and promoter-sequence determinants for initial-transcription pausing

Time-resolved cryoEM using Spotiton

Contact Info

Product

Resources

About