Single molecule analysis reveals reversible and irreversible steps during spliceosome activation

Hoskins, Aaron A.; Rodgers, Margaret L.; Friedman, Larry J.; Gelles, Jeff; Moore, Melissa J.

doi:10.7554/elife.14166

Cited by 42 publications

(56 citation statements)

References 54 publications

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“…Photobleaching contributions for σ 70 RNAP 647 dwell times on rrnB P1 DNA and GreB Cy3B on surface-tethered σ 70 RNAP or RNAP core determined as described previously (Hoskins et al, 2016) and were negligible. Tables Table 1. From single-or bi-exponential fit.…”

Section: Cosmos Data Analysismentioning

confidence: 99%

Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription

Stumper

Ravi

Friedman

et al. 2019

eLife

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RNA polymerases (RNAPs) contain a conserved ‘secondary channel’ which binds regulatory factors that modulate transcription initiation. In Escherichia coli, the secondary channel factors (SCFs) GreB and DksA both repress ribosomal RNA (rRNA) transcription, but SCF loading and repression mechanisms are unclear. We observed in vitro fluorescently labeled GreB molecules binding to single RNAPs and initiation of individual transcripts from an rRNA promoter. GreB arrived and departed from promoters only in complex with RNAP. GreB did not alter initial RNAP-promoter binding but instead blocked a step after conformational rearrangement of the initial RNAP-promoter complex. Strikingly, GreB-RNAP complexes never initiated at an rRNA promoter; only RNAP molecules arriving at the promoter without bound GreB produced transcript. The data reveal that a model SCF functions by a ‘delayed inhibition’ mechanism and suggest that rRNA promoters are inhibited by GreB/DksA because their short-lived RNAP complexes do not allow sufficient time for SCFs to dissociate.

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Section: Cosmos Data Analysismentioning

confidence: 99%

Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription

Stumper

Ravi

Friedman

et al. 2019

eLife

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“…Regulatory factors or regions, including the CC and CC-binding proteins, may modulate the kinetics of U4/U6 unwinding by Brr2, which could tune the velocity of Bact formation. In addition, Brr2 lacking the Nterminal region can lead to non-canonical disruption of the U4/U6•U5 tri-snRNP (into U4/U6 di-snRNP and U5 snRNP (28), and B complex spliceosomes have been shown to undergo repeated attempts at activation with intermittent release of U4 (67). Thus, Brr2 might be able to remodel the B complex in diverse ways, and thereby differentially funnel B complexes based on different pre-mRNA substrates along or off the splicing pathway.…”

Section: Implications For Splicing Regulationmentioning

confidence: 99%

Positive and negative intra-molecular modulation in a dual-cassette RNA helicase

Vester

Kuropka

et al. 2019

Preprint

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RNA helicase Brr2 is required for the activation of the spliceosome prior to the first catalytic step of splicing. Brr2 represents a distinct subgroup of Ski2-like nucleic acid helicases whose members comprise tandem helicase cassettes. Only the Nterminal cassette of Brr2 is an active ATPase and can unwind substrate RNAs. The C-terminal cassette represents a pseudo-enzyme that can stimulate RNA-related activities of the N-terminal cassette. However, the molecular mechanisms, by which the C-terminal cassette modulates the activities of the N-terminal unit remain elusive. Here, we show that N-and C-terminal cassettes adopt vastly different relative orientations in a crystal structure of Brr2 in complex with an activating domain of the spliceosomal Prp8 protein as compared to the crystal structure of isolated Brr2. Likewise, the cassettes occupy different relative positions and engage in different intercassette contacts during different stages of splicing. Engineered disulfide bridges that lock the cassettes in two different relative orientations have opposite effects on RNA-related activities of the N-terminal cassette compared to the unrestrained protein.Moreover, different relative positioning of the cassettes strongly influences ATP hydrolysis by the N-terminal cassette. Our results demonstrate that the inactive C-terminal cassette of Brr2 can exert both positive and negative influence on the active N-terminal helicase unit from a distance. Medicine,

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“…However, single molecule FRET experiments performed in yeast whole cell extracts show that each step of spliceosome assembly is reversible; the chances of an individual intron being spliced increase as the spliceosome successfully completes each assembly step[78]. Because of the heterogeneity inherent in In vitro splicing reactions, single molecule techniques are essential to our understanding of reversibility as well as other dynamic changes in the spliceosome.…”

Section: 0 Before the Structuresmentioning

confidence: 99%

Genetics and biochemistry remain essential in the structural era of the spliceosome

Mayerle

Guthrie

2017

Methods

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The spliceosome is not a single macromolecular machine. Rather it is a collection of dynamic heterogeneous subcomplexes that rapidly interconvert throughout the course of a typical splicing cycle. Because of this, for many years the only high resolution structures of the spliceosome available were of smaller, isolated protein or RNA components. Consequently much of our current understanding of the spliceosome derives from biochemical and genetic techniques. Now with the publication of multiple, high resolution structures of the spliceosome, some question the relevance of traditional biochemical and genetic techniques to the splicing field. We argue such techniques are not only relevant, but vital for an in depth mechanistic understanding of pre-mRNA splicing.

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Single molecule analysis reveals reversible and irreversible steps during spliceosome activation

Cited by 42 publications

References 54 publications

Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription

Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription

Positive and negative intra-molecular modulation in a dual-cassette RNA helicase

Genetics and biochemistry remain essential in the structural era of the spliceosome

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