Structural visualization of de novo transcription initiation by Saccharomyces cerevisiae RNA polymerase II

Yang, Chun Li; Fujiwara, Rina; Kim, Hee Jong; Basnet, Pratik; Zhu, Yunye; Colón, Jose J. Gorbea; Steimle, Stefan; Garcia, Benjamin A.; Kaplan, Craig D.; Murakami, Kenji

doi:10.1016/j.molcel.2021.12.020

Cited by 13 publications

(10 citation statements)

References 96 publications

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“…More supporting evidence comes from a comparison of the mammalian ITC structures with the structure of the yeast ITC on a G-less promoter (G is at +26) ( 47 ) (fig. S6 and supplementary text).…”

Section: Resultsmentioning

confidence: 99%

“…Although structures of reconstituted ITCs have been reported (40)(41)(42)(43)(44)(45)(46), these complexes were not started from a PIC but instead were assembled on artificial RNA-DNA hybrids that were formed by a mismatched transcription bubble in which the template strand paired with a synthetic complementary RNA. A recent study captured a structure of a yeast (Saccharomyces cerevisiae) backtracked ITC complex that started from a PIC (47). However, transcription initiation in yeast and mammals differs in various aspects.…”

Section: Structural Visualization Of Transcription Initiation In Actionmentioning

confidence: 99%

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Structural visualization of transcription initiation in action

Chen,

Liu,

Wang

et al. 2023

Science

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Transcription initiation is a complex process, and its mechanism is incompletely understood. We determined the structures of de novo transcribing complexes TC2 to TC17 with RNA polymerase II halted on G-less promoters when nascent RNAs reach 2 to 17 nucleotides in length, respectively. Connecting these structures generated a movie and a working model. As initially synthesized RNA grows, general transcription factors (GTFs) remain bound to the promoter and the transcription bubble expands. Nucleoside triphosphate (NTP)–driven RNA-DNA translocation and template-strand accumulation in a nearly sealed channel may promote the transition from initially transcribing complexes (ITCs) (TC2 to TC9) to early elongation complexes (EECs) (TC10 to TC17). Our study shows dynamic processes of transcription initiation and reveals why ITCs require GTFs and bubble expansion for initial RNA synthesis, whereas EECs need GTF dissociation from the promoter and bubble collapse for promoter escape.

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Section: Resultsmentioning

confidence: 99%

Section: Structural Visualization Of Transcription Initiation In Actionmentioning

confidence: 99%

Structural visualization of transcription initiation in action

Chen,

Liu,

Wang

et al. 2023

Science

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“…Although it was previously reported that both the N-term and C-term of Tfb3 are essential for viability in S. cerevisiae (Warfield et al, 2016), we found that cells lacking the C-terminal domain are viable but extremely slow growing. Interestingly, accurate transcription initiation could be reconstituted in vitro using a Tfb3 truncation consisting of only N-term residues 1-148 (Yang et al, 2022). However, we found that adding back the C-terminal domain as a separate protein dramatically improved growth ( Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, yeast transcription reconstituted in vitro with TFIIH lacking Tfb3 and the Kinase Module causes a dramatic upstream shift in transcription start sites, with the TATA-TSS spacing now resembling that of metazoans (Murakami et al, 2015). This effect can be reversed by adding back only the Tfb3 N-term (Yang et al, 2022), arguing against a model where the contact between Tfb3 and Ssl2 is necessary for downstream TSS usage. Nevertheless, there is clearly some communication between the Kinase and Core Modules, and Tfb3 is likely to be a key link.…”

Section: Discussionmentioning

confidence: 99%

Uncoupling the TFIIH Core and Kinase Modules Leads To Misregulated RNA Polymerase II CTD Serine 5 Phosphorylation

Giordano,

Buratowski,

Jeronimo

et al. 2023

Preprint

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TFIIH is an essential transcription initiation factor for RNA polymerase II (RNApII). This multi-subunit complex comprises two modules that are physically linked by the subunit Tfb3 (MAT1 in metazoans). The TFIIH Core Module, with two DNA-dependent ATPases and several additional subunits, promotes DNA unwinding. The TFIIH Kinase Module phosphorylates Serine 5 of the C-terminal domain (CTD) of RNApII subunit Rpb1, a modification that coordinates exchange of initiation and early elongation factors. While it is not obvious why these two disparate activities are bundled into one factor, the connection may provide temporal coordination during early initiation. Here we show that Tfb3 can be split into two parts to uncouple the TFIIH modules. The resulting cells grow slower than normal, but are viable. Chromatin immunoprecipitation of the split TFIIH shows that the Core Module, but not the Kinase, is properly recruited to promoters. Instead of the normal promoter-proximal peak, high CTD Serine 5 phosphorylation is seen throughout transcribed regions. Therefore, coupling the TFIIH modules is necessary to localize and limit CTD kinase activity to early stages of transcription. These results are consistent with the idea that the two TFIIH modules began as independent functional entities that became connected by Tfb3 during early eukaryotic evolution.

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“…To study the mechanisms of promoter escape, an efficient in vitro system for de novo , promoter-dependent transcription is required that allows for reconstitution of the initiation- elongation transition in the test tube and for subsequent structural characterization of the transition intermediates. Two recent studies reported such in vitro transcription initiation systems for the yeast Saccharomyces cerevisiae 27,28 . However, the initiation-elongation transition differs between the yeast and human systems.…”

Section: Introductionmentioning

confidence: 99%

Three-step mechanism of promoter escape by RNA polymerase II

Zhan,

Grabbe,

Oberbeckmann

et al. 2023

Preprint

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SUMMARYThe transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how interactions between RNA polymerase (Pol) II and initiation factors are broken to enable promoter escape. Here we reconstitute Pol II promoter escapein vitro, determine high-resolution structures of five transition intermediates, and show that promoter escape occurs in three major steps. First, the growing RNA transcript displaces the B-reader element of the initiation factor TFIIB without evicting TFIIB. Second, rewinding of the upstream edge of the growing DNA bubble evicts TFIIA, TFIIB and TBP and repositions parts of TFIIE and TFIIF. Third, binding of DSIF and NELF evicts TFIIE and TFIIH, establishing the paused elongation complex. This three-step model for promoter escape fills a gap in our understanding of the initiation-elongation transition of Pol II transcription.

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Structural visualization of de novo transcription initiation by Saccharomyces cerevisiae RNA polymerase II

Cited by 13 publications

References 96 publications

Structural visualization of transcription initiation in action

Structural visualization of transcription initiation in action

Uncoupling the TFIIH Core and Kinase Modules Leads To Misregulated RNA Polymerase II CTD Serine 5 Phosphorylation

Three-step mechanism of promoter escape by RNA polymerase II

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