RNA Polymerase II Transcription
About: This article is published in Journal of Molecular Biology.The article was published on 2021-05-03 and is currently open access. It has received 2 citations till now. The article focuses on the topics: RNA polymerase II & Transcription (biology).
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TL;DR: In this article , RNA polymerase II-associated protein 2 (RPAP2) was identified to transport Pol II from cytoplasm to nucleus and dephosphorylates Pol II C-terminal domain (CTD) with undetectable phosphatase activity.
4 citations
TL;DR: In this paper, RNA polymerase II-associated protein 2 (RPAP2) was identified to transport Pol II from cytoplasm to nucleus and dephosphorylates Pol II C-terminal domain (CTD).
Abstract: RNA polymerase II (Pol II)-mediated transcription in metazoan requires precise regulation. RNA polymerase II-associated protein 2 (RPAP2) was previously identified to transport Pol II from cytoplasm to nucleus and dephosphorylates Pol II C-terminal domain (CTD). We found that RPAP2 binds hypo/hyper-phosphorylated Pol II with undetectable phosphatase activity. Structure of RPAP2-Pol II shows mutually exclusive assembly of RPAP2-Pol II and pre-initiation complex (PIC) due to three steric clashes. RPAP2 prevents/disrupts Pol II-TFIIF interaction and impairs in vitro transcription initiation, suggesting a function in prohibiting PIC assembly. Loss of RPAP2 in cells leads to global accumulation of TFIIF and Pol II at promoters, indicating critical role of RPAP2 in inhibiting PIC assembly independent of its putative phosphatase activity. Our study indicates that RPAP2 functions as a gatekeeper to prohibit PIC assembly and transcription initiation and suggests a novel transcription checkpoint.
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References
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TL;DR: In this paper, the authors identify 21 meta-assemblages consisting of roughly 400 different proteins that are related to DNA replication, centromeres, subtelomeres, transposons and transcription by RNA polymerase (Pol) I, II and III.
Abstract: The genome-wide architecture of chromatin-associated proteins that maintains chromosome integrity and gene regulation is not well defined. Here we use chromatin immunoprecipitation, exonuclease digestion and DNA sequencing (ChIP–exo/seq)1,2 to define this architecture in Saccharomyces cerevisiae. We identify 21 meta-assemblages consisting of roughly 400 different proteins that are related to DNA replication, centromeres, subtelomeres, transposons and transcription by RNA polymerase (Pol) I, II and III. Replication proteins engulf a nucleosome, centromeres lack a nucleosome, and repressive proteins encompass three nucleosomes at subtelomeric X-elements. We find that most promoters associated with Pol II evolved to lack a regulatory region, having only a core promoter. These constitutive promoters comprise a short nucleosome-free region (NFR) adjacent to a +1 nucleosome, which together bind the transcription-initiation factor TFIID to form a preinitiation complex. Positioned insulators protect core promoters from upstream events. A small fraction of promoters evolved an architecture for inducibility, whereby sequence-specific transcription factors (ssTFs) create a nucleosome-depleted region (NDR) that is distinct from an NFR. We describe structural interactions among ssTFs, their cognate cofactors and the genome. These interactions include the nucleosomal and transcriptional regulators RPD3-L, SAGA, NuA4, Tup1, Mediator and SWI–SNF. Surprisingly, we do not detect interactions between ssTFs and TFIID, suggesting that such interactions do not stably occur. Our model for gene induction involves ssTFs, cofactors and general factors such as TBP and TFIIB, but not TFIID. By contrast, constitutive transcription involves TFIID but not ssTFs engaged with their cofactors. From this, we define a highly integrated network of gene regulation by ssTFs. A ChIP–exo method is used to define the genome-wide positional organization of proteins associated with gene transcription, DNA replication, centromeres, subtelomeres and transposons, revealing distinct protein assemblies for constitutive and inducible gene expression.
84 citations
TL;DR: In this article, the authors present the structure of the human Mediator-bound preinitiation complex (PIC) at a resolution below 4 angstroms, where the coactivator Mediator is recruited by transcription factors, facilitates the assembly of the PIC, and stimulates phosphorylation of the Pol II C-terminal domain (CTD) by the TFIIH subunit CDK7.
Abstract: Eukaryotic transcription requires the assembly of a multisubunit preinitiation complex (PIC) composed of RNA polymerase II (Pol II) and the general transcription factors. The coactivator Mediator is recruited by transcription factors, facilitates the assembly of the PIC, and stimulates phosphorylation of the Pol II C-terminal domain (CTD) by the TFIIH subunit CDK7. Here, we present the cryo-electron microscopy structure of the human Mediator-bound PIC at a resolution below 4 angstroms. Transcription factor binding sites within Mediator are primarily flexibly tethered to the tail module. CDK7 is stabilized by multiple contacts with Mediator. Two binding sites exist for the Pol II CTD, one between the head and middle modules of Mediator and the other in the active site of CDK7, providing structural evidence for Pol II CTD phosphorylation within the Mediator-bound PIC.
68 citations
TL;DR: In this paper, a recombinant version of human Mediator, reconstitute a 50-subunit Mediator-PIC complex and determine the structure of the complex by cryo-electron microscopy.
Abstract: Mediator is a conserved coactivator complex that enables the regulated initiation of transcription at eukaryotic genes1–3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate the phosphorylation of RNA polymerase II (Pol II) and promoter escape1–6. Here we prepare a recombinant version of human Mediator, reconstitute a 50-subunit Mediator–PIC complex and determine the structure of the complex by cryo-electron microscopy. The head module of Mediator contacts the stalk of Pol II and the general transcription factors TFIIB and TFIIE, resembling the Mediator–PIC interactions observed in the corresponding complex in yeast7–9. The metazoan subunits MED27–MED30 associate with exposed regions in MED14 and MED17 to form the proximal part of the Mediator tail module that binds activators. Mediator positions the flexibly linked cyclin-dependent kinase (CDK)-activating kinase of the general transcription factor TFIIH near the linker to the C-terminal repeat domain of Pol II. The Mediator shoulder domain holds the CDK-activating kinase subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook domains reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of the CDK7 kinase to allosterically stimulate phosphorylation of the C-terminal domain. The structure of a recombinant 20-subunit version of human Mediator bound to the transcription pre-initiation complex is determined, providing insight into the regulation of RNA polymerase II initiation.
55 citations
TL;DR: A host of proteins required for the disassembly, reassembly, and modification of nucleosomes interacts with RNA polymerase II to aid its movement and counteract its disruptive effects on chromatin this paper.
40 citations
TL;DR: The transcription elongation factor Spt5 is conserved from bacteria to humans and has been implicated in both gene-specific regulation and transcription through nucleosomes and its inhibitory and enhancing activities in transcription.
34 citations