Characterizing Single‐Molecule FRET Dynamics with Probability Distribution Analysis

Santoso, Yusdi; Torella, Joseph P.; Kapanidis, Achillefs N.

doi:10.1002/cphc.201000129

Cited by 76 publications

(95 citation statements)

References 49 publications

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“…A possible explanation for the wide distribution of the RP o FRET peak is the presence of conformational heterogeneity in RP o, 26,33,34 which may exist in multiple static conformational states that do not interconvert within the millisecond-timescale transit of single RP o complexes through the detection volume. Alternatively, RP o may be dynamic and interconvert between different conformational states within the millisecond timescale.…”

Section: Resultsmentioning

confidence: 99%

“…Specifically, we have performed single-molecule FRET ( F örster r esonance e nergy t ransfer) (smFRET) measurements on individual, freely diffusing molecules of RP o in solution 24,26–28 in order to detect transcription-bubble flexibility, to distinguish between static and dynamic transcription-bubble flexibility and to relate transcription-bubble flexibility and dynamics to start-site selection. By measuring the FRET efficiency between fluorophore pairs probing different regions within promoter DNA, we were able to detect the formation of the transcription bubble (Fig.…”

Section: Introductionmentioning

confidence: 99%

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The Transcription Bubble of the RNA Polymerase–Promoter Open Complex Exhibits Conformational Heterogeneity and Millisecond-Scale Dynamics: Implications for Transcription Start-Site Selection

Robb

Cordes

Hwang

et al. 2013

Journal of Molecular Biology

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Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 bp around the transcription start site and forms a single-stranded “transcription bubble” within a catalytically active RNAP–DNA open complex (RPo). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5′ end of RNA transcripts and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RPo. The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion (“scrunching”) or bubble contraction (“unscrunching”). Here, we assess the presence of dynamic flexibility in RPo with single-molecule FRET (Förster resonance energy transfer). We obtain experimental evidence for dynamic flexibility in RPo using different FRET rulers and labeling positions. An analysis of FRET distributions of RPo using burst variance analysis reveals conformational fluctuations in RPo in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RPo. Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RPo and indicates that DNA dynamics within the bubble affect the search for transcription start sites.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The Transcription Bubble of the RNA Polymerase–Promoter Open Complex Exhibits Conformational Heterogeneity and Millisecond-Scale Dynamics: Implications for Transcription Start-Site Selection

Robb

Cordes

Hwang

et al. 2013

Journal of Molecular Biology

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“…A custom-built confocal microscope was used for smFRET experiments as previously described (45)(46)(47), and the setup was modified to allow ALEX of donor and acceptor fluorophores (26,48). Increasing concentrations of viral RNAP were incubated with 1 nM ATTO647N-labeled RNA for 15 min at 28°C in 50 mM Tris·HCl (pH 8), 500 mM NaCl, 10 mM MgCl 2 , 100 μg/μL BSA, 1 mM DTT, and 5% glycerol before being diluted in a buffer comprising 50 mM Tris·HCl (pH 8), 100 mM KCl, 10 mM MgCl 2 , 100 μg/μL BSA, 1 mM DTT, and 5% glycerol to a final RNA concentration of 100 pM for confocal analysis.…”

Section: Methodsmentioning

confidence: 99%

Single-molecule FRET reveals a corkscrew RNA structure for the polymerase-bound influenza virus promoter

Tomescu

Robb

Hengrung

et al. 2014

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

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The influenza virus is a major human and animal pathogen responsible for seasonal epidemics and occasional pandemics. The genome of the influenza A virus comprises eight segments of singlestranded, negative-sense RNA with highly conserved 5′ and 3′ termini. These termini interact to form a double-stranded promoter structure that is recognized and bound by the viral RNA-dependent RNA polymerase (RNAP); however, no 3D structural information for the influenza polymerase-bound promoter exists. Functional studies have led to the proposal of several 2D models for the secondary structure of the bound promoter, including a corkscrew model in which the 5′ and 3′ termini form short hairpins. We have taken advantage of an insect-cell system to prepare large amounts of active recombinant influenza virus RNAP, and used this to develop a highly sensitive single-molecule FRET assay to measure distances between fluorescent dyes located on the promoter and map its structure both with and without the polymerase bound. These advances enabled the direct analysis of the influenza promoter structure in complex with the viral RNAP, and provided 3D structural information that is in agreement with the corkscrew model for the influenza virus promoter RNA. Our data provide insights into the mechanisms of promoter binding by the influenza RNAP and have implications for the understanding of the regulatory mechanisms involved in the transcription of viral genes and replication of the viral RNA genome. In addition, the simplicity of this system should translate readily to the study of any virus polymerase-promoter interaction.

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“…Date of recurrence was defined as an oncologist-confirmed return of cancer after treatment and remission, abstracted directly from each patient's electronic medical record 20 .…”

Section: Study Cohortmentioning

confidence: 99%

Cost and Resource Utilization in Cervical Cancer Management: A Real-World Retrospective Cost Analysis

et al. 2016

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ObjectivesWe set out to assess the health care resource utilization and cost of cervical cancer from the perspective of a single-payer health care system. MethodsRetrospective observational data for women diagnosed with cervical cancer in British Columbia between 2004 and 2009 were analyzed to calculate patient-level resource utilization patterns from diagnosis to death or 5-year discharge. Domains of resource use within the scope of this cost analysis were chemotherapy, radiotherapy, and brachytherapy administered by the BC Cancer Agency; resource utilization related to hospitalization and outpatient visits as recorded by the B.C. Ministry of Health; medically required services billed under the B.C. Medical Services Plan; and prescriptions dispensed under British Columbia's health insurance programs. Unit costs were applied to radiotherapy and brachytherapy, producing per-patient costs. ResultsThe mean cost per case of treating cervical cancer in British Columbia was $19,153 (standard error: $3,484).Inpatient hospitalizations, at 35%, represented the largest proportion of the total cost (95% confidence interval: 32.9% to 36.9%). Costs were compared for subgroups of the total cohort. ConclusionsAs health care systems change the way they manage, screen for, and prevent cervical cancer, costeffectiveness evaluations of the overall approach will require up-to-date data for resource utilization and costs. We provide information suitable for such a purpose and also identify factors that influence costs.

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Characterizing Single‐Molecule FRET Dynamics with Probability Distribution Analysis

Cited by 76 publications

References 49 publications

The Transcription Bubble of the RNA Polymerase–Promoter Open Complex Exhibits Conformational Heterogeneity and Millisecond-Scale Dynamics: Implications for Transcription Start-Site Selection

The Transcription Bubble of the RNA Polymerase–Promoter Open Complex Exhibits Conformational Heterogeneity and Millisecond-Scale Dynamics: Implications for Transcription Start-Site Selection

Single-molecule FRET reveals a corkscrew RNA structure for the polymerase-bound influenza virus promoter

Cost and Resource Utilization in Cervical Cancer Management: A Real-World Retrospective Cost Analysis

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