Time will tell: comparing timescales to gain insight into transcriptional bursting

Trends in Genetics, Journal Year: 2024, Volume and Issue: 40(2), P. 160 - 174

Published: Jan. 12, 2024

Recent imaging studies have captured the dynamics of regulatory events transcription inside living cells. These include factor (TF) DNA binding, chromatin remodeling and modification, enhancer-promoter (E-P) proximity, cluster formation, preinitiation complex (PIC) assembly. Together, these molecular culminate in stochastic bursts RNA synthesis, but their kinetic relationship remains largely unclear. In this review, we compare timescales upstream steps (input) with kinetics transcriptional bursting (output) to generate mechanistic models single We highlight open questions potential technical advances guide future endeavors toward a quantitative understanding regulation.

Language: Английский

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Real-time visualization of reconstituted transcription reveals RNA polymerase II activation mechanisms at single promoters

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 6, 2025

RNA polymerase II (RNAPII) is regulated by sequence-specific transcription factors (TFs) and the pre-initiation complex (PIC): TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, Mediator. TFs Mediator contain intrinsically-disordered regions (IDRs) form phase-separated condensates, but how IDRs control RNAPII function remains poorly understood. Using purified PIC factors, we developed a Real-time In-vitro Fluorescence Transcription assay (RIFT) for second-by-second visualization of at hundreds promoters simultaneously. We show rapid activation IDR-dependent, without condensate formation. For example, MED1-IDR can functionally replace native TF, activating with similar (not identical) kinetics; however, squelches as condensate, activates single-protein. cooperatively activate bursting re-initiation surprisingly, drive TF-promoter recruitment, TF-DNA binding. Collectively, RIFT addressed questions largely intractable cell-based methods, yielding mechanistic insights about IDRs, enhancer-promoter communication, that complement live-cell imaging data.

Language: Английский

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Dynamic microenvironments shape nuclear organization and gene expression

Current Opinion in Genetics & Development, Journal Year: 2024, Volume and Issue: 86, P. 102177 - 102177

Published: March 11, 2024

Language: Английский

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Live-cell single-molecule dynamics of eukaryotic RNA polymerase machineries

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: July 11, 2024

Abstract Eukaryotic gene expression in the nucleus is orchestrated by three RNA polymerases (RNAP-I, -II, and -III) associated factors 1,2 . Despite extensive biochemical, genomic, structural, imaging studies, real-time dynamics of these transcription complexes remain obscure. Here, we employ single-molecule tracking living yeast to assess physiological kinetics over 50 representative proteins encompassing all RNAP machineries. Components RNAPI RNAPIII pre-initiation (PICs) engage long-lived interactions on chromatin, reflecting their roles for constitutive rRNA tRNA synthesis, contrast transient RNAPII PIC. We further report key components across cycle 2–5 —factors upstream regulation, elongation, histone modification, C-terminal domain (CTD) processing, termination—revealing unprecedented insights into temporal landscape transcription. Strikingly, many elongation factors, previously thought travel processively with RNAPII, display residence times, suggesting highly dynamic rather than constant association. Systematic screening RNAPII-associated shows that truncation RNAPII-CTD substantially reduces U1 snRNP time decreases intron retention ribosomal protein genes, providing how CTD length influences co-transcriptional splicing. Our findings establish a framework chromatin polymerase machineries cells.

Language: Английский

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Promoter and Gene-Body RNA-Polymerase II co-exist in partial demixed condensates

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: March 17, 2024

In cells, transcription is tightly regulated on multiple layers. The condensation of the machinery into distinct phases hypothesised to spatio-temporally fine tune RNA polymerase II behaviour during two key stages, initiation and elongation nascent transcripts. However, it has remained unclear whether these would mix when present at same time or remain chemical environments; either as multi-phase condensates by forming entirely separate condensates. Here we combine particle-based multi-scale simulations experiments in model organism C. elegans characterise biophysical properties Both vivo work describe a lower critical solution temperature (LCST) Polymerase II, with dissolving temperatures whereas higher promote condensate stability. Importantly this gradual change correlates an incremental transcriptional response temperature, but largely uncoupled from classical stress response. LCST CTD also highlights that are physio-chemically heterochromatin Expanding how degree phosphorylation disordered C-terminal domain (CTD), which characteristic for each step transcription, controls demixing pCTD line phase separation experiments. We show putatively underpinning constitute environments agreement observed embryos super resolution microscopy. Our analysis reveals depending its post-translational modifications interaction partners single protein can adopt morphologies partially engulfed selective recruitment additional factors different phases.

Language: Английский

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Distinct Mechanisms of Recognition of Phosphorylated RNAPII C-Terminal Domain by BRCT Repeats of the BRCA1-BARD1 Complex: Insights from Structural and Functional Analyses

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 22, 2025

Abstract Transcription competes with other DNA-dependent processes, such as DNA repair, for access to its substrate, DNA. However, the principles governing interplay between these processes remain poorly understood. Evidence suggests that BRCA1-BARD1 complex, a key player in damage response, may act mediator of this crosstalk. In study, we investigated molecular mechanism underpinning interaction RNA polymerase II (RNAPII) and well functional implications. Our findings reveal BRCT repeat BRCA1 binds Ser5-phosphorylated CTD RNAPII, utilising previously established ligands. Furthermore, demonstrate is critical organisation RNAPII into condensates liquid-like properties. Analysis disease-associated variants within repeats further supports biological relevance condensation. Collectively, our results suggest complex coordinate transcription repair by facilitating factories.

Language: Английский

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RNA polymerase II partitioning is a shared feature of diverse oncofusion condensates

Cell, Journal Year: 2025, Volume and Issue: unknown

Published: April 1, 2025

Language: Английский

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Regulation of RNA polymerase II transcription through re-initiation and bursting

Molecular Cell, Journal Year: 2025, Volume and Issue: 85(10), P. 1907 - 1919

Published: May 1, 2025

Language: Английский

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Molecular basis of global promoter sensing and nucleosome capture by the SWR1 chromatin remodeler

Cell, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 1, 2024

Language: Английский

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Nonspecific Interactions in Transcription Regulation and Organization of Transcriptional Condensates

Biochemistry (Moscow), Journal Year: 2024, Volume and Issue: 89(4), P. 688 - 700

Published: April 1, 2024

Language: Английский

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