Learning Rates and States from Biophysical Time Series: A Bayesian Approach to Model Selection and Single-Molecule FRET Data

Bronson, Jonathan E.; Fei, Jingyi; Hofman, Jake M.; Gonzalez, Ruben L.; Wiggins, Chris H.

doi:10.1016/j.bpj.2009.09.031

Cited by 393 publications

(527 citation statements)

References 39 publications

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“…Data were analyzed with custom MATLAB (The Mathworks) image-processing software. A three E FRETstate model was fit with program vbFRET (30). See SI Materials and Methods for more details.…”

Section: Methodsmentioning

confidence: 99%

Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5′ splice site and branch site

Crawford

Hoskins

Friedman

et al. 2013

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

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Removal of introns from the precursors to messenger RNA (premRNAs) requires close apposition of intron ends by the spliceosome, but when and how apposition occurs is unclear. We investigated the process by which intron ends are brought together using single-molecule fluorescence resonance energy transfer together with colocalization single-molecule spectroscopy, a combination of methods that can directly reveal how conformational transitions in macromolecular machines are coupled to specific assembly and disassembly events. The FRET measurements suggest that the 5′ splice site and branch site remain physically separated throughout spliceosome assembly, and only approach one another after the spliceosome is activated for catalysis, at which time the pre-mRNA becomes highly dynamic. Separation of the sites of chemistry until very late in the splicing pathway may be crucial for preventing splicing at incorrect sites.splicing mechanism | single-molecule FRET | RNA dynamics I ntron excision from precursors to messenger RNAs (premRNAs) is carried out by the spliceosome, arguably the most complex macromolecular machine in the cell (1). One of the most important jobs of the spliceosome is to accurately and efficiently identify the ends of introns and bring them together to promote the chemistry of splicing. This chemistry occurs via two S N 2 transesterification reactions: (i) attack by the branch site (BS) adenosine on the phosphodiester bond at the beginning of the intron (the 5′ splice site; 5′SS) and (ii) attack of the released 5′ exon on the phosphodiester bond at the end of the intron (the 3′SS) (2). The BS adenosine is internal to the intron and usually located in the vicinity of the 3′SS.The spliceosome consists of four major subcomplexes that must assemble de novo on each new intron: the U1 and U2 small nuclear ribonucleoprotein particles (snRNPs), the U4/U6.U5 tri-snRNP, and the protein-only nineteen complex (NTC). The snRNPs each contain numerous proteins and one or more small nuclear RNAs (snRNAs). These subcomplexes assemble stepwise, with U1 and U2 recognition of the 5′SS and BS, respectively, preceding trisnRNP and NTC recruitment (3, 4). Throughout the assembly process, numerous large-scale conformational changes occur that involve making and breaking of pre-mRNA:snRNA and snRNA: snRNA base pairing interactions. These structural transitions are necessary for both recognition of the splice sites and creation of the catalytic core in which the splice sites are juxtaposed for chemistry. The two chemical steps occur within the activated spliceosome formed after ejection of the U1 and U4 snRNPs (5).When during spliceosome assembly are the splice sites brought into close proximity? Previous studies in human and yeast extracts using hydroxy radical cleavage or protein-RNA crosslinking led to the hypothesis that the 5′SS and BS regions are closely positioned in early complexes containing only U1 and/or U2 (6-12). However, as both of these irreversible trapping methods can capture transient excursions that are no...

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“…Data were analyzed with custom MATLAB (The Mathworks) image-processing software. A three E FRETstate model was fit with program vbFRET (30). See SI Materials and Methods for more details.…”

Section: Methodsmentioning

confidence: 99%

Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5′ splice site and branch site

Crawford

Hoskins

Friedman

et al. 2013

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

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“…The fluorescence intensity changes (Figs. 1B and 2A) were detected statistically using a hidden Markov model, analyzed with the variational Bayes method (38).…”

Section: Methodsmentioning

confidence: 99%

Dynamically varying interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells

Hiroshima

Saeki

Okada‐Hatakeyama

et al. 2012

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

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Heregulin (HRG) belongs to the family of EGFs and activates the receptor proteins ErbB3 and ErbB4 in a variety of cell types to regulate cell fate. The interactions between HRG and ErbB3/B4 are important to the pathological mechanisms underlying schizophrenia and some cancers. Here, we observed the reaction kinetics between fluorescently labeled single HRG molecules and ErbB3/B4 on the surfaces of MCF-7 human breast cancer cells. The equilibrium association and the dissociation from equilibrium were also measured using single-molecule imaging techniques. The unitary association processes mirrored the EGF and ErbB1 interactions in HeLa cells [Teramura Y, et al. (2006) , suggesting that the predimerization of the receptors, followed by intermediate formation (between the first and second ligand-binding events to a receptor dimer), accelerated the formation of doubly liganded signaling dimers of the receptor molecules. However, the dissociation analysis suggested that the first HRG dissociation from the doubly liganded dimer was rapid, but the second dissociation from the singly liganded dimer was slow. The dissociation rate constant from the liganded monomer was intermediate. The dynamic changes in the association and dissociation kinetics in relation to the dimerization of ErbB displayed negative cooperativity, which resulted in apparent low-and high-affinity sites of HRG association on the cell surface.ligand-receptor interaction | cell signaling | epidermal growth factors T he ErbB family includes four member proteins, which localize to the plasma membrane of various types of cells and mediate signal transduction involved in cell differentiation and proliferation (1, 2). Among the ErbB family members, ErbB3 and ErbB4 are receptors for several extracellular protein ligands, including heregulin (HRG, also called neuregulin). ErbB3 is involved in the development of the heart and nervous system, and ErbB4 plays a role in lactation (3, 4). The dysregulation of ErbB3 and B4 (referred to hereafter as "HRG receptors") is often related to malignancy in human cancers (2). The long-term exposure of MCF-7 cells, a cultured cell line derived from a human breast carcinoma, to HRG induces the production of differentiation markers, such as milk proteins or lipid droplets, whereas similar exposure to epidermal growth factor induces proliferation (5).ErbB molecules must dimerize to transduce signals across the plasma membrane (3,4,6). Single ErbB molecules associate with single ligand molecules, such as HRG, and two liganded receptor molecules form a doubly liganded dimer on the plasma membrane (7). Dimerization is essential for the activation of ErbB molecules. This activation involves the phosphorylation of multiple sites along the cytoplasmic tail domain, which is catalyzed by the cytoplasmic kinase domain of the dimerization partner. In addition to homodimerization, some combinations of heterodimerization between members of the ErbB family have been described (1, 2). Several forms of unliganded and liganded homoand heterodi...

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“…By using this approach, we could achieve a fluorescence intensity of approximately 100 photons ms −1 , which is sufficient for 0.3 ms resolution on the FRET time trace (Figure 3). We selected time traces longer than 8 ms and then applied a hidden Markov model (HMM) analysis to the time traces 18. Figure 3 a shows typical time traces for R40S30 with a 0.3 ms temporal resolution.…”

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confidence: 99%

Dynamic Release of Bending Stress in Short dsDNA by Formation of a Kink and Forks

et al. 2015

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Bending with high curvature is one of the major mechanical properties of double‐stranded DNA (dsDNA) that is essential for its biological functions. The emergence of a kink arising from local melting in the middle of dsDNA has been suggested as a mechanism of releasing the energy cost of bending. Herein, we report that strong bending induces two types of short dsDNA deformations, induced by two types of local melting, namely, a kink in the middle and forks at the ends, which we demonstrate using D‐shaped DNA nanostructures. The two types of deformed dsDNA structures dynamically interconvert on a millisecond timescale. The transition from a fork to a kink is dominated by entropic contribution (anti‐Arrhenius behavior), while the transition from a kink to a fork is dominated by enthalpic contributions. The presence of mismatches in dsDNA accelerates kink formation, and the transition from a kink to a fork is removed when the mismatch size is three base pairs.

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Learning Rates and States from Biophysical Time Series: A Bayesian Approach to Model Selection and Single-Molecule FRET Data

Cited by 393 publications

References 39 publications

Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5′ splice site and branch site

Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5′ splice site and branch site

Dynamically varying interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells

Dynamic Release of Bending Stress in Short dsDNA by Formation of a Kink and Forks

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