scholarly journals Cotranscriptional R-loop formation by Mfd involves topological partitioning of DNA

2021 ◽  
Vol 118 (15) ◽  
pp. e2019630118
Author(s):  
James R. Portman ◽  
Gwendolyn M. Brouwer ◽  
Jack Bollins ◽  
Nigel J. Savery ◽  
Terence R. Strick
Keyword(s):  
Nucleic Acid ◽  
Rna Polymerase ◽  
Real Time ◽  
Single Molecule ◽  
Loop Formation ◽  
Regulatory Processes ◽  
Template Sequence ◽  
Gene Regulatory ◽  
Nascent Rna ◽  
Positive Supercoiling

R-loops are nucleic acid hybrids which form when an RNA invades duplex DNA to pair with its template sequence. Although they are implicated in a growing number of gene regulatory processes, their mechanistic origins remain unclear. We here report real-time observations of cotranscriptional R-loop formation at single-molecule resolution and propose a mechanism for their formation. We show that the bacterial Mfd protein can simultaneously interact with both elongating RNA polymerase and upstream DNA, tethering the two together and partitioning the DNA into distinct supercoiled domains. A highly negatively supercoiled domain forms in between Mfd and RNA polymerase, and compensatory positive supercoiling appears in front of the RNA polymerase and behind Mfd. The nascent RNA invades the negatively supercoiled domain and forms a stable R-loop that can drive mutagenesis. This mechanism theoretically enables any protein that simultaneously binds an actively translocating RNA polymerase and upstream DNA to stimulate R-loop formation.

scholarly journals Non-denaturing bisulfite treatment and single-molecule real-time sequencing reveals d-loop footprints, their length, position and distribution

2020 ◽  
Author(s):  
Shanaya Shital Shah ◽  
Stella Hartono ◽  
Frédéric Chédin ◽  
Wolf-Dietrich Heyer
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Detection Methods ◽  
Loop Formation ◽  
Bisulfite Treatment ◽  
Loop Detection ◽  
D Loop ◽  
Donor Choice ◽  
The Relationship

ABSTRACTDisplacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of a crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loop with base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint, revealing D-loop characteristics at unprecedented resolution. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.

scholarly journals Single-Stranded DNA Curtains for Real-Time Single-Molecule Visualization of Protein–Nucleic Acid Interactions

2012 ◽  
Vol 84 (18) ◽  
pp. 7607-7612 ◽  
Author(s):  
Bryan Gibb ◽  
Tim D. Silverstein ◽  
Ilya J. Finkelstein ◽  
Eric C. Greene
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Single Molecule ◽  
Single Stranded Dna ◽  
Protein Nucleic Acid ◽  
Nucleic Acid Interactions

scholarly journals Single-Molecule Imaging of RNA Polymerase-DNA Interactions in Real Time

1999 ◽  
Vol 76 (2) ◽  
pp. 709-715 ◽  
Author(s):  
Yoshie Harada ◽  
Takashi Funatsu ◽  
Katsuhiko Murakami ◽  
Yoshikazu Nonoyama ◽  
Akira Ishihama ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Real Time ◽  
Single Molecule ◽  
Single Molecule Imaging ◽  
Dna Interactions

Real-time monitoring of cotranscriptional riboswitch folding and switching

2019 ◽  
Author(s):  
Boyang Hua ◽  
Christopher P. Jones ◽  
Jaba Mitra ◽  
Peter J. Murray ◽  
Rebecca Rosenthal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Real Time ◽  
Single Molecule ◽  
Rna Folding ◽  
Its Sequence ◽  
Transcriptional Terminator ◽  
Fate Control ◽  
New Methods ◽  
Nascent Chain

SummaryRiboswitches function through cotranscriptional conformation switching governed by cognate ligand concentration, RNA folding and transcription elongation kinetics. To investigate how these parameters influence riboswitch folding, we developed a novel vectorial folding assay (VF) in which the superhelicase Rep-X sequentially liberates the RNA strand from a heteroduplex in a 5’-to-3’ direction, mimicking the nascent chain emergence during transcription. The RNA polymerase (RNAP)-free VF recapitulates the kinetically controlled cotranscriptional folding of a ZTP riboswitch, whose activation is favored by slower transcription, strategic pausing, or a weakened transcriptional terminator. New methods to observe positions and local rates of individual helicases show an average Rep-X unwinding rate similar to bacterial RNAP elongation (~60 nt/s). Real-time single-molecule monitoring captured folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These methods allow observation of individual folding RNAs as they occupy distinct folding channels within the landscape that controls gene expression and showed that riboswitch fate control is encoded in its sequence and is readily interpreted by a directionally moving protein even in the absence of an RNA polymerase.

scholarly journals Single-Stranded DNA Curtains for Real-Time Single-Molecule Visualization of Protein−Nucleic Acid Interactions

2013 ◽  
Vol 104 (2) ◽  
pp. 178a
Author(s):  
Bryan Gibb ◽  
Tim D. Silverstein ◽  
Ilya J. Finkelstein ◽  
Eric C. Greene
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Single Molecule ◽  
Single Stranded Dna ◽  
Protein Nucleic Acid ◽  
Nucleic Acid Interactions

scholarly journals Real-time single-cell characterization of the eukaryotic transcription cycle reveals correlations between RNA initiation, elongation, and cleavage

2021 ◽  
Vol 17 (5) ◽  
pp. e1008999
Author(s):  
Jonathan Liu ◽  
Donald Hansen ◽  
Elizabeth Eck ◽  
Yang Joon Kim ◽  
Meghan Turner ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Single Cell ◽  
Real Time ◽  
Fruit Fly ◽  
Effective Parameters ◽  
Spatiotemporal Resolution ◽  
Eukaryotic Transcription ◽  
Nascent Rna ◽  
Transcription Cycle

The eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript. Although each of these steps can be regulated as well as coupled with each other, their in vivo dissection has remained challenging because available experimental readouts lack sufficient spatiotemporal resolution to separate the contributions from each of these steps. Here, we describe a novel application of Bayesian inference techniques to simultaneously infer the effective parameters of the transcription cycle in real time and at the single-cell level using a two-color MS2/PP7 reporter gene and the developing fruit fly embryo as a case study. Our method enables detailed investigations into cell-to-cell variability in transcription-cycle parameters as well as single-cell correlations between these parameters. These measurements, combined with theoretical modeling, suggest a substantial variability in the elongation rate of individual RNA polymerase molecules. We further illustrate the power of this technique by uncovering a novel mechanistic connection between RNA polymerase density and nascent RNA cleavage efficiency. Thus, our approach makes it possible to shed light on the regulatory mechanisms in play during each step of the transcription cycle in individual, living cells at high spatiotemporal resolution.

scholarly journals Single-molecule real-time transcript sequencing identified flowering regulatory genes in Crocus sativus

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Xiaodong Qian ◽  
Youping Sun ◽  
Guifen Zhou ◽  
Yumei Yuan ◽  
Jing Li ◽  
...  
Keyword(s):  
Real Time ◽  
Gene Regulatory Network ◽  
Single Molecule ◽  
Regulatory Network ◽  
Cold Treatment ◽  
Regulatory Genes ◽  
Full Length ◽  
Crocus Sativus ◽  
Medicinal Uses ◽  
Gene Regulatory

Abstract Background Saffron crocus (Crocus sativus) is a valuable spice with medicinal uses in gynaecopathia and nervous system diseases. Identify flowering regulatory genes plays a vital role in increasing flower numbers, thereby resulting in high saffron yield. Results Two full length transcriptome gene sets of flowering and non-flowering saffron crocus were established separately using the single-molecule real-time (SMRT) sequencing method. A total of sixteen SMRT cells generated 22.85 GB data and 75,351 full-length saffron crocus unigenes on the PacBio RS II panel and further obtained 79,028 SSRs, 72,603 lncRNAs and 25,400 alternative splicing (AS) events. Using an Illumina RNA-seq platform, an additional fifteen corms with different flower numbers were sequenced. Many differential expression unigenes (DEGs) were screened separately between flowering and matched non-flowering top buds with cold treatment (1677), flowering top buds of 20 g corms and non-flowering top buds of 6 g corms (1086), and flowering and matched non-flowering lateral buds (267). A total of 62 putative flower-related genes that played important roles in vernalization (VRNs), gibberellins (G3OX, G2OX), photoperiod (PHYB, TEM1, PIF4), autonomous (FCA) and age (SPLs) pathways were identified and a schematic representation of the flowering gene regulatory network in saffron crocus was reported for the first time. After validation by real-time qPCR in 30 samples, two novel genes, PB.20221.2 (p = 0.004, r = 0.52) and PB.38952.1 (p = 0.023, r = 0.41), showed significantly higher expression levels in flowering plants. Tissue distribution showed specifically high expression in flower organs and time course expression analysis suggested that the transcripts increasingly accumulated during the flower development period. Conclusions Full-length transcriptomes of flowering and non-flowering saffron crocus were obtained using a combined NGS short-read and SMRT long-read sequencing approach. This report is the first to describe the flowering gene regulatory network of saffron crocus and establishes a reference full-length transcriptome for future studies on saffron crocus and other Iridaceae plants.

scholarly journals Real-time monitoring of single ZTP riboswitches reveals a complex and kinetically controlled decision landscape

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Boyang Hua ◽  
Christopher P. Jones ◽  
Jaba Mitra ◽  
Peter J. Murray ◽  
Rebecca Rosenthal ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Rna Polymerase ◽  
Real Time ◽  
Single Molecule ◽  
Rna Folding ◽  
Transcription Terminator ◽  
Kinetically Controlled ◽  
Bacterial Rna ◽  
Stress Sensing ◽  
And Function

Abstract RNAs begin to fold and function during transcription. Riboswitches undergo cotranscriptional switching in the context of transcription elongation, RNA folding, and ligand binding. To investigate how these processes jointly modulate the function of the folate stress-sensing Fusobacterium ulcerans ZTP riboswitch, we apply a single-molecule vectorial folding (VF) assay in which an engineered superhelicase Rep-X sequentially releases fluorescently labeled riboswitch RNA from a heteroduplex in a 5′-to-3′ direction, at ~60 nt s−1 [comparable to the speed of bacterial RNA polymerase (RNAP)]. We demonstrate that the ZTP riboswitch is kinetically controlled and that its activation is favored by slower unwinding, strategic pausing between but not before key folding elements, or a weakened transcription terminator. Real-time single-molecule monitoring captures folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These results show how individual nascent RNAs occupy distinct channels within the folding landscape that controls the fate of the riboswitch.

Single-Molecule, Real-Time Dissecting of Peptide Nucleic Acid–DNA Duplexes with a Protein Nanopore Tweezer

2018 ◽  
Vol 90 (12) ◽  
pp. 7682-7690 ◽  
Author(s):  
Andrei Ciuca ◽  
Alina Asandei ◽  
Irina Schiopu ◽  
Aurelia Apetrei ◽  
Loredana Mereuta ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Single Molecule ◽  
Peptide Nucleic Acid ◽  
Dna Duplexes

scholarly journals Bisulfite treatment and single-molecule real-time sequencing reveal D-loop length, position, and distribution

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Shanaya Shital Shah ◽  
Stella R Hartono ◽  
Frédéric Chédin ◽  
Wolf-Dietrich Heyer
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Detection Methods ◽  
Loop Formation ◽  
Vitro Assay ◽  
Bisulfite Treatment ◽  
Loop Detection ◽  
D Loop ◽  
Donor Choice

Displacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loops with near base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.

Sign in / Sign up

Export Citation Format

Share Document