Real-time observation of the initiation of RNA polymerase II transcription

Fazal, Furqan M.; Meng, Cong A.; Murakami, Kenji; Kornberg, Roger D.; Block, Steven M.

doi:10.1038/nature14882

Cited by 103 publications

(83 citation statements)

References 38 publications

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“…However, during early elongation, tight contacts with promoter DNA and GTFs often persist, preventing the polymerase from moving forward along DNA as the RNA chain is extended and causing "scrunching" of an extended transcription bubble within Pol II (Fig. 1C;Fazal et al 2015). This conformation is energetically unfavorable, and, after >10 nt of RNA has been formed, the upstream region of the extended bubble collapses (Holstege et al 1997;Hieb et al 2006;Luse 2013), allowing template and nontemplate DNA to reassociate.…”

Section: Pausing Takes Center Stage In Gene Regulationmentioning

confidence: 99%

Promoter-proximal pausing of RNA polymerase II: a nexus of gene regulation

2019

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Precise spatio-temporal control of gene activity is essential for organismal development, growth, and survival in a changing environment. Decisive steps in gene regulation involve the pausing of RNA polymerase II (Pol II) in early elongation, and the controlled release of paused polymerase into productive RNA synthesis. Here we describe the factors that enable pausing and the events that trigger Pol II release into the gene. We also discuss open questions in the field concerning the stability of paused Pol II, nucleosomes as obstacles to elongation, and potential roles of pausing in defining the precision and dynamics of gene expression.Cell type-and condition-specific patterns of gene expression involve tight control of multiple steps in the transcription cycle. Early regulatory events govern the association of DNA-binding transcription factors (TFs) with their target motifs and the recruitment of RNA polymerase II (Pol II) and general TFs (GTFs) to the promoter region of a gene. These events enable transcription initiation and the synthesis of a short, nascent RNA. However, Pol II then pauses promoter-proximally, awaiting further signals before entering the gene body. The fate of the paused early elongation complex is absolutely decisive for gene output. If paused Pol II successfully transitions into productive elongation, a functional full-length mRNA can be made. However, a failure in the maturation of paused Pol II toward productive RNA synthesis short circuits the process of gene expression. Thus, understanding the factors that govern stable Pol II pausing and release are of paramount importance toward an appreciation of gene regulation in human health and disease.

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Section: Pausing Takes Center Stage In Gene Regulationmentioning

confidence: 99%

Promoter-proximal pausing of RNA polymerase II: a nexus of gene regulation

2019

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“…However, scrunching in TSS selection occurs before NTP binding and nucleotide incorporation and, in the absence of an additional energy source, is driven by energy available from the thermal bath and therefore limited to ~1–3 bp--rather than occurring after NTP binding and nucleotide incorporation, being driven by a combination of thermal energy, NTP binding, and nucleotide incorporation, and being able to span tens of bp. We suggest that, in the presence of an additional energy source, scrunching in TSS selection could access a larger range of TSS positions, and, in particular, we speculate that this occurs with TFIIH-dependent ATP hydrolysis as the additional energy source in the long-range “TSS scanning” observed with eukaryotic RNAPII in some species (17). …”

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confidence: 95%

Multiplexed protein-DNA cross-linking: Scrunching in transcription start site selection

Winkelman

Vvedenskaya

Zhang

et al. 2016

Science

107

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In bacterial transcription initiation, RNA polymerase (RNAP) selects a transcription start site (TSS) at variable distances downstream of core promoter elements. Using next-generation sequencing and unnatural-amino-acid-mediated protein-DNA crosslinking, we have determined, for a library of 410 promoter sequences, the TSS, the RNAP leading-edge position, and the RNAP trailing-edge position. We find that a promoter element upstream of the TSS, the “discriminator,” participates in TSS selection, and that, as the TSS changes, the RNAP leading-edge position changes, but the RNAP trailing-edge position does not change. Changes in the RNAP leading-edge position but not the RNAP trailing-edge position are a defining hallmark of the “DNA scrunching” that occurs concurrent with RNA synthesis in initial transcription. We propose that TSS selection involves DNA scrunching prior to RNA synthesis.

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“…Ssl2 promotes formation of the Pol II open complex by reeling downstream promoter DNA into the Pol II active site, leading to DNA unwinding (11,12). In budding yeast, Pol II transcription initiates downstream from the site of PIC formation (13), and Ssl2 function drives the scanning of downstream promoter DNA for an appropriate TSS (14). In the human transcription system, XPB activity has been implicated in promoter escape and the release of Pol II from contacts with promoter DNA and other PIC components (15,16).…”

mentioning

confidence: 99%

Function of Conserved Topological Regions within the Saccharomyces cerevisiae Basal Transcription Factor TFIIH

Warfield

Luo

Ranish

et al. 2016

Molecular and Cellular Biology

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bTFIIH is a 10-subunit RNA polymerase II basal transcription factor with a dual role in DNA repair. TFIIH contains three enzymatic functions and over 30 conserved subdomains and topological regions. We systematically tested the function of these regions in three TFIIH core module subunits, i.e., Ssl1, Tfb4, and Tfb2, in the DNA translocase subunit Ssl2, and in the kinase module subunit Tfb3. Our results are consistent with previously predicted roles for the Tfb2 Hub, Ssl2 Lock, and Tfb3 Latch regions, with mutations in these elements typically having severe defects in TFIIH subunit association. We also found unexpected roles for other domains whose function had not previously been defined. First, the Ssl1-Tfb4 Ring domains are important for TFIIH assembly. Second, the Tfb2 Hub and HEAT domains have an unexpected role in association with Tfb3. Third, the Tfb3 Ring domain is important for association with many other TFIIH subunits. Fourth, a partial deletion of the Ssl1 N-terminal extension (NTE) domain inhibits TFIIH function without affecting subunit association. Finally, we used site-specific cross-linking to localize the Tfb3-binding surface on the Rad3 Arch domain. Our cross-linking results suggest that Tfb3 and Rad3 have an unusual interface, with Tfb3 binding on two opposite faces of the Arch. TFIIH is a conserved 10-subunit complex that plays essential roles in both RNA polymerase II (Pol II) transcription and nucleotide excision repair (NER) (1-3). TFIIH and TFIIE are the last basal factors to join the transcription preinitiation complex (PIC) (4). In transcription, TFIIH functions in DNA unwinding, transcription start site (TSS) scanning, promoter escape, and Pol II C-terminal domain (CTD) phosphorylation. During NER, TFIIH promotes the DNA unwinding of an asymmetric region around the site of DNA lesions as an early step in excision and replacement of damaged DNA (5, 6). Mutations in three TFIIH subunits, XPD, XPB, and p8, cause human diseases associated with defects in DNA repair and transcription (1).TFIIH has three enzymatic activities that are essential for these functions. First, the Rad3/XPD (yeast/human) subunit is an ATPdependent DNA helicase that participates in DNA unwinding during NER. While this enzymatic activity is dispensable for transcription, Rad3/XPD is required for transcription as it plays an essential structural role in stabilizing the TFIIH complex (7-10). Second, Ssl2/XPB is an ATP-dependent double-stranded DNA translocase with roles in transcription and DNA repair. Ssl2 promotes formation of the Pol II open complex by reeling downstream promoter DNA into the Pol II active site, leading to DNA unwinding (11, 12). In budding yeast, Pol II transcription initiates downstream from the site of PIC formation (13), and Ssl2 function drives the scanning of downstream promoter DNA for an appropriate TSS (14). In the human transcription system, XPB activity has been implicated in promoter escape and the release of Pol II from contacts with promoter DNA and other PIC components (15, 16). Ssl2...

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Real-time observation of the initiation of RNA polymerase II transcription

Cited by 103 publications

References 38 publications

Promoter-proximal pausing of RNA polymerase II: a nexus of gene regulation

Promoter-proximal pausing of RNA polymerase II: a nexus of gene regulation

Multiplexed protein-DNA cross-linking: Scrunching in transcription start site selection

Function of Conserved Topological Regions within the Saccharomyces cerevisiae Basal Transcription Factor TFIIH

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