scholarly journals Structure of transcribing RNA polymerase II-nucleosome complex

2018 ◽  
Author(s):  
Lucas Farnung ◽  
Seychelle M. Vos ◽  
Patrick Cramer
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Published Data ◽  
Elongation Factors ◽  
Dna Templates ◽  
Protein Coding ◽  
Pol Ii ◽  
Nucleosome Core ◽  
Mechanistic Analysis ◽  
Starting Point

Transcription of eukaryotic protein-coding genes requires passage of RNA polymerase II (Pol II) through chromatin. Pol II passage is impaired by nucleosomes and requires elongation factors that help Pol II to efficiently overcome the nucleosomal barrier1-4. How the Pol II machinery transcribes through a nucleosome remains unclear because structural studies have been limited to Pol II elongation complexes formed on DNA templates lacking nucleosomes5. Here we report the cryo-electron microscopy (cryo-EM) structure of transcribing Pol II from the yeast Saccharomyces cerevisiae engaged with a downstream nucleosome core particle (NCP) at an overall resolution of 4.4 Å with resolutions ranging from 4-6 Å in Pol II and 6-8 Å in the NCP. Pol II and the NCP adopt a defined orientation that could not be predicted from modelling. Pol II contacts DNA of the incoming NCP on both sides of the nucleosomal dyad with its domains ‘clamp head’ and ‘lobe’. Comparison of the Pol II-NCP structure to known structures of Pol II complexes reveals that the elongation factors TFIIS, DSIF, NELF, PAF1 complex, and SPT6 can be accommodated on the Pol II surface in the presence of the oriented nucleosome. Further structural comparisons show that the chromatin remodelling enzyme Chd1, which is also required for efficient Pol II passage6,7, could bind the oriented nucleosome with its motor domain. The DNA-binding region of Chd1 must however be released from DNA when Pol II approaches the nucleosome, and based on published data8,9 this is predicted to stimulate Chd1 activity and to facilitate Pol II passage. Our results provide a starting point for a mechanistic analysis of chromatin transcription.

scholarly journals cis- and trans-Acting Determinants of Transcription Termination by Yeast RNA Polymerase II

2006 ◽  
Vol 26 (7) ◽  
pp. 2688-2696 ◽  
Author(s):  
Eric J. Steinmetz ◽  
Sarah B. H. Ng ◽  
Joseph P. Cloute ◽  
David A. Brow
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Nascent Transcript ◽  
Protein Coding ◽  
Pol Ii ◽  
Eukaryotic Genes ◽  
Discrete Surface ◽  
Cleavage And Polyadenylation ◽  
Selection For

ABSTRACT Most eukaryotic genes are transcribed by RNA polymerase II (Pol II), including those that produce mRNAs and many noncoding functional RNAs. Proper expression of these genes requires efficient termination by Pol II to avoid transcriptional interference and synthesis of extended, nonfunctional RNAs. We previously described a pathway for yeast Pol II termination that involves recognition of an element in the nascent transcript by the essential RNA-binding protein Nrd1. The Nrd1-dependent pathway appears to be used primarily for nonpolyadenylated transcripts, such as the small nuclear and small nucleolar RNAs (snoRNAs). mRNAs are thought to use a distinct pathway that is coupled to cleavage and polyadenylation of the transcript. Here we show that the terminator elements for two yeast snoRNA genes also direct polyadenylated 3′-end formation in the context of an mRNA 3′ untranslated region. A selection for cis-acting terminator readthrough mutations identified conserved features of these elements, some of which are similar to cleavage and polyadenylation signals. A selection for trans-acting mutations that induce readthrough of both a snoRNA and an mRNA terminator yielded mutations in the Rpb3 and Rpb11 subunits of Pol II that define a remarkably discrete surface on the trailing end of the enzyme. Our results suggest that, at least in budding yeast, protein-coding and noncoding Pol II-transcribed genes use similar mechanisms to direct termination and that the termination signal is transduced through the Rpb3/Rpb11 heterodimer.

scholarly journals CDK9 and PP2A regulate the link between RNA polymerase II transcription termination and RNA maturation.

2021 ◽  
Author(s):  
Michael Tellier ◽  
Justyna Zaborowska ◽  
Jonathan Neve ◽  
Takayuki Nojima ◽  
Svenja Hester ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Premature Termination ◽  
Transcription Termination ◽  
Elongation Factor ◽  
Protein Coding ◽  
Pol Ii ◽  
Working Together ◽  
Rna Maturation ◽  
Nascent Transcripts

CDK9 is a critical kinase required for the productive transcription of protein-coding genes by RNA polymerase II (pol II) in higher eukaryotes. Phosphorylation of targets including the elongation factor SPT5 and the carboxyl-terminal domain (CTD) of RNA pol II allows the polymerase to pass an early elongation checkpoint (EEC), which is encountered soon after initiation. In addition to halting RNA polymerase II at the EEC, CDK9 inhibition also causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, suggesting that CDK9 and PP2A, working together, regulate the coupling of elongation and transcription termination to RNA maturation. Our phosphoproteomic analyses, using either DRB or an ATP analog-sensitive CDK9 cell line confirm the splicing factor SF3B1 as an additional key target of this kinase. CDK9 inhibition causes loss of interaction of splicing and export factors with SF3B1, suggesting that CDK9 also helps to co-ordinates coupling of splicing and export to transcription.

scholarly journals RNA polymerase II clustering through CTD phase separation

2018 ◽  
Author(s):  
M. Boehning ◽  
C. Dugast-Darzacq ◽  
M. Rankovic ◽  
A. S. Hansen ◽  
T. Yu ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Low Complexity ◽  
Initiation Factor ◽  
Published Data ◽  
Pol Ii ◽  
Intrinsically Disordered ◽  
Ctd Phosphorylation

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation at increasing protein concentration, with the shorter yeast CTD forming less stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters/hubs at active genes through interactions between CTDs and with activators, and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.

scholarly journals Structure of a transcribing RNA polymerase II–U1 snRNP complex

Science ◽  
2021 ◽  
Vol 371 (6526) ◽  
pp. 305-309 ◽  
Author(s):  
Suyang Zhang ◽  
Shintaro Aibara ◽  
Seychelle M. Vos ◽  
Dmitry E. Agafonov ◽  
Reinhard Lührmann ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Splice Site ◽  
Messenger Rna ◽  
Loop Formation ◽  
Mrna Isoforms ◽  
Pol Ii ◽  
Small Nuclear Ribonucleoprotein ◽  
Starting Point ◽  
U1 Snrnp

To initiate cotranscriptional splicing, RNA polymerase II (Pol II) recruits the U1 small nuclear ribonucleoprotein particle (U1 snRNP) to nascent precursor messenger RNA (pre-mRNA). Here, we report the cryo–electron microscopy structure of a mammalian transcribing Pol II–U1 snRNP complex. The structure reveals that Pol II and U1 snRNP interact directly. This interaction positions the pre-mRNA 5′ splice site near the RNA exit site of Pol II. Extension of pre-mRNA retains the 5′ splice site, leading to the formation of a “growing intron loop.” Loop formation may facilitate scanning of nascent pre-mRNA for the 3′ splice site, functional pairing of distant intron ends, and prespliceosome assembly. Our results provide a starting point for a mechanistic analysis of cotranscriptional spliceosome assembly and the biogenesis of mRNA isoforms by alternative splicing.

scholarly journals Regulation of expression of human RNA polymerase II-transcribed snRNA genes

Open Biology ◽  
2017 ◽  
Vol 7 (6) ◽  
pp. 170073 ◽  
Author(s):  
Joana Guiro ◽  
Shona Murphy
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Molecular Mechanisms ◽  
Mrna Splicing ◽  
Regulation Of Expression ◽  
Protein Coding ◽  
Pol Ii ◽  
Protein Coding Genes ◽  
Non Coding Rnas ◽  
Rna Genes

In addition to protein-coding genes, RNA polymerase II (pol II) transcribes numerous genes for non-coding RNAs, including the small-nuclear (sn)RNA genes. snRNAs are an important class of non-coding RNAs, several of which are involved in pre-mRNA splicing. The molecular mechanisms underlying expression of human pol II-transcribed snRNA genes are less well characterized than for protein-coding genes and there are important differences in expression of these two gene types. Here, we review the DNA features and proteins required for efficient transcription of snRNA genes and co-transcriptional 3′ end formation of the transcripts.

scholarly journals Different phosphoisoforms of RNA polymerase II engage the Rtt103 termination factor in a structurally analogous manner

2017 ◽  
Vol 114 (20) ◽  
pp. E3944-E3953 ◽  
Author(s):  
Corey M. Nemec ◽  
Fan Yang ◽  
Joshua M. Gilmore ◽  
Corinna Hintermair ◽  
Yi-Hsuan Ho ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Protein Complexes ◽  
Transcription Termination ◽  
Structural Data ◽  
Side Chain ◽  
Protein Coding ◽  
Pol Ii ◽  
Carboxyl Terminal ◽  
Nucleolar Rna

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestrates dynamic recruitment of specific cellular machines during different stages of transcription. Signature phosphorylation patterns of Y1S2P3T4S5P6S7 heptapeptide repeats of the CTD engage specific “readers.” Whereas phospho-Ser5 and phospho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only impact specific genes. Here, we identify a role for phospho-Thr4 in transcription termination at noncoding small nucleolar RNA (snoRNA) genes. Quantitative proteomics reveals an interactome of known readers as well as protein complexes that were not known to rely on Thr4 for association with Pol II. The data indicate a key role for Thr4 in engaging the machinery used for transcription elongation and termination. We focus on Rtt103, a protein that binds phospho-Ser2 and phospho-Thr4 marks and facilitates transcription termination at protein-coding genes. To elucidate how Rtt103 engages two distinct CTD modifications that are differentially enriched at noncoding genes, we relied on NMR analysis of Rtt103 in complex with phospho-Thr4– or phospho-Ser2–bearing CTD peptides. The structural data reveal that Rtt103 interacts with phospho-Thr4 in a manner analogous to its interaction with phospho-Ser2–modified CTD. The same set of hydrogen bonds involving either the oxygen on phospho-Thr4 and the hydroxyl on Ser2, or the phosphate on Ser2 and the Thr4 hydroxyl, can be formed by rotation of an arginine side chain, leaving the intermolecular interface otherwise unperturbed. This economy of design enables Rtt103 to engage Pol II at distinct sets of genes with differentially enriched CTD marks.

Role of the C-terminal domain of RNA polymerase II in expression of small nuclear RNA genes

2008 ◽  
Vol 36 (3) ◽  
pp. 537-539 ◽  
Author(s):  
Sylvain Egloff ◽  
Shona Murphy
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Repeat Unit ◽  
Small Nuclear Rna ◽  
Protein Coding ◽  
Pol Ii ◽  
Terminal Domain ◽  
Covalent Modifications

Pol II (RNA polymerase II) transcribes the genes encoding proteins and non-coding snRNAs (small nuclear RNAs). The largest subunit of Pol II contains a distinctive CTD (C-terminal domain) comprising a repetitive heptad amino acid sequence, Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. This domain is now known to play a major role in the processes of transcription and co-transcriptional RNA processing in expression of both snRNA and protein-coding genes. The heptapeptide repeat unit can be extensively modified in vivo and covalent modifications of the CTD during the transcription cycle result in the ordered recruitment of RNA-processing factors. The most studied modifications are the phosphorylation of the serine residues in position 2 and 5 (Ser2 and Ser5), which play an important role in the co-transcriptional processing of both mRNA and snRNA. An additional, recently identified CTD modification, phosphorylation of the serine residue in position 7 (Ser7) of the heptapeptide, is however specifically required for expression of snRNA genes. These findings provide interesting insights into the control of gene-specific Pol II function.

scholarly journals HSV-1 ICP22 Is a Selective Viral Repressor of Cellular RNA Polymerase II-Mediated Transcription Elongation

Vaccines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1054
Author(s):  
Nur Firdaus Isa ◽  
Olivier Bensaude ◽  
Nadiah C. Aziz ◽  
Shona Murphy
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Viral Gene ◽  
Elongation Factors ◽  
Pol Ii ◽  
Early Protein ◽  
Hsv 1

The Herpes Simplex Virus (HSV-1) immediate-early protein ICP22 interacts with cellular proteins to inhibit host cell gene expression and promote viral gene expression. ICP22 inhibits phosphorylation of Ser2 of the RNA polymerase II (pol II) carboxyl-terminal domain (CTD) and productive elongation of pol II. Here we show that ICP22 affects elongation of pol II through both the early-elongation checkpoint and the poly(A)-associated elongation checkpoint of a protein-coding gene model. Coimmunoprecipitation assays using tagged ICP22 expressed in human cells and pulldown assays with recombinant ICP22 in vitro coupled with mass spectrometry identify transcription elongation factors, including P-TEFb, additional CTD kinases and the FACT complex as interacting cellular factors. Using a photoreactive amino acid incorporated into ICP22, we found that L191, Y230 and C225 crosslink to both subunits of the FACT complex in cells. Our findings indicate that ICP22 interacts with critical elongation regulators to inhibit transcription elongation of cellular genes, which may be vital for HSV-1 pathogenesis. We also show that the HSV viral activator, VP16, has a region of structural similarity to the ICP22 region that interacts with elongation factors, suggesting a model where VP16 competes with ICP22 to deliver elongation factors to viral genes.

scholarly journals HSV-1 ICP22 is a selective viral repressor of cellular RNA polymerase II-mediated transcription elongation

2021 ◽  
Author(s):  
Nur Firdaus Isa ◽  
Olivier Bensaude ◽  
Nadiah C. Aziz ◽  
Shona Murphy
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Viral Gene ◽  
Elongation Factors ◽  
Pol Ii ◽  
Early Protein ◽  
Hsv 1

The Herpes Simplex Virus (HSV-1) immediate early protein ICP22 interacts with cellular proteins to inhibit host cell gene expression and promote viral gene expression. ICP22 inhibits phosphorylation of Ser2 of the RNA polymerase II (pol II) carboxyl-terminal domain (CTD) and productive elongation of pol II. Here we show that ICP22 affects elongation of pol II through both the early-elongation checkpoint and the poly(A)-associated elongation checkpoint on a protein-coding gene model. Coimmunoprecipitation assays using tagged ICP22 expressed in human cells and pulldown assays with recombinant ICP22 in vitro coupled with mass spectrometry identify transcription elongation factors, including P-TEFb, additional CTD kinases and the FACT complex as interacting cellular factors. Using a photoreactive amino acid incorporated into ICP22, we found that L191, Y230 and C225 crosslink to both subunits of the FACT complex in cells.  Our findings indicate that ICP22 physically interacts with critical elongation regulators to inhibit transcription elongation of cellular genes, which may be vital for HSV-1 pathogenesis. We also show that the HSV viral activator, VP16 has a region of structural similarity to the ICP22 region that interacts with elongation factors, suggesting a model where VP16 competes with ICP22 to deliver elongation factors to viral genes.

scholarly journals Opposite roles of transcription elongation factors Spt4/5 and Elf1 in RNA polymerase II transcription through B-form versus non-B DNA structures

2021 ◽  
Author(s):  
Jun Xu ◽  
Jenny Chong ◽  
Dong Wang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Trinucleotide Repeat ◽  
Transcription Elongation ◽  
Dna Lesions ◽  
Cag Repeat ◽  
Elongation Factors ◽  
Pol Ii ◽  
Dna Structures

Abstract Transcription elongation can be affected by numerous types of obstacles, such as nucleosome, pausing sequences, DNA lesions and non-B-form DNA structures. Spt4/5 and Elf1 are conserved transcription elongation factors that promote RNA polymerase II (Pol II) bypass of nucleosome and pausing sequences. Importantly, genetic studies have shown that Spt4/5 plays essential roles in the transcription of expanded nucleotide repeat genes associated with inherited neurological diseases. Here, we investigate the function of Spt4/5 and Elf1 in the transcription elongation of CTG•CAG repeat using an in vitro reconstituted yeast transcription system. We found that Spt4/5 helps Pol II transcribe through the CTG•CAG tract duplex DNA, which is in good agreement with its canonical roles in stimulating transcription elongation. In sharp contrast, surprisingly, we revealed that Spt4/5 greatly inhibits Pol II transcriptional bypass of CTG and CAG slip-out structures. Furthermore, we demonstrated that transcription elongation factor Elf1 individually and cooperatively with Spt4/5 inhibits Pol II bypass of the slip-out structures. This study uncovers the important functional interplays between template DNA structures and the function of transcription elongation factors. This study also expands our understanding of the functions of Spt4/5 and Elf1 in transcriptional processing of trinucleotide repeat DNA.

Sign in / Sign up

Export Citation Format

Share Document