Publications by authors named "Lucas Farnung"

15 Publications

  • Page 1 of 1

Structural basis of nucleosome transcription mediated by Chd1 and FACT.

Nat Struct Mol Biol 2021 Apr 12;28(4):382-387. Epub 2021 Apr 12.

Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Göttingen, Germany.

Efficient transcription of RNA polymerase II (Pol II) through nucleosomes requires the help of various factors. Here we show biochemically that Pol II transcription through a nucleosome is facilitated by the chromatin remodeler Chd1 and the histone chaperone FACT when the elongation factors Spt4/5 and TFIIS are present. We report cryo-EM structures of transcribing Saccharomyces cerevisiae Pol II-Spt4/5-nucleosome complexes with bound Chd1 or FACT. In the first structure, Pol II transcription exposes the proximal histone H2A-H2B dimer that is bound by Spt5. Pol II has also released the inhibitory DNA-binding region of Chd1 that is poised to pump DNA toward Pol II. In the second structure, Pol II has generated a partially unraveled nucleosome that binds FACT, which excludes Chd1 and Spt5. These results suggest that Pol II progression through a nucleosome activates Chd1, enables FACT binding and eventually triggers transfer of FACT together with histones to upstream DNA.
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http://dx.doi.org/10.1038/s41594-021-00578-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046669PMC
April 2021

Mechanism of SARS-CoV-2 polymerase stalling by remdesivir.

Nat Commun 2021 01 12;12(1):279. Epub 2021 Jan 12.

Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Am Fassberg 11, Göttingen, 37077, Germany.

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3'-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3'-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3'-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.
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http://dx.doi.org/10.1038/s41467-020-20542-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7804290PMC
January 2021

Nucleosome-CHD4 chromatin remodeler structure maps human disease mutations.

Elife 2020 06 16;9. Epub 2020 Jun 16.

Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Göttingen, Germany.

Chromatin remodeling plays important roles in gene regulation during development, differentiation and in disease. The chromatin remodeling enzyme CHD4 is a component of the NuRD and ChAHP complexes that are involved in gene repression. Here, we report the cryo-electron microscopy (cryo-EM) structure of CHD4 engaged with a nucleosome core particle in the presence of the non-hydrolysable ATP analogue AMP-PNP at an overall resolution of 3.1 Å. The ATPase motor of CHD4 binds and distorts nucleosomal DNA at superhelical location (SHL) +2, supporting the 'twist defect' model of chromatin remodeling. CHD4 does not induce unwrapping of terminal DNA, in contrast to its homologue Chd1, which functions in gene activation. Our structure also maps CHD4 mutations that are associated with human cancer or the intellectual disability disorder Sifrim-Hitz-Weiss syndrome.
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http://dx.doi.org/10.7554/eLife.56178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338049PMC
June 2020

Structure of complete Pol II-DSIF-PAF-SPT6 transcription complex reveals RTF1 allosteric activation.

Nat Struct Mol Biol 2020 07 15;27(7):668-677. Epub 2020 Jun 15.

Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Göttingen, Germany.

Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation factors DSIF, PAF1 complex (PAF) and SPT6. Here we report the cryo-EM structure of the complete EC* that contains RTF1, a dissociable PAF subunit critical for chromatin transcription. The RTF1 Plus3 domain associates with Pol II subunit RPB12 and the phosphorylated C-terminal region of DSIF subunit SPT5. RTF1 also forms four α-helices that extend from the Plus3 domain along the Pol II protrusion and RPB10 to the polymerase funnel. The C-terminal 'fastener' helix retains PAF and is followed by a 'latch' that reaches the end of the bridge helix, a flexible element of the Pol II active site. RTF1 strongly stimulates Pol II elongation, and this requires the latch, possibly suggesting that RTF1 activates transcription allosterically by influencing Pol II translocation.
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http://dx.doi.org/10.1038/s41594-020-0437-1DOI Listing
July 2020

Structure of replicating SARS-CoV-2 polymerase.

Nature 2020 08 21;584(7819):154-156. Epub 2020 May 21.

Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

The new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. Here we present a cryo-electron microscopy structure of the SARS-CoV-2 RdRp in an active form that mimics the replicating enzyme. The structure comprises the viral proteins non-structural protein 12 (nsp12), nsp8 and nsp7, and more than two turns of RNA template-product duplex. The active-site cleft of nsp12 binds to the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the second turn of RNA. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged 'sliding poles'. These sliding poles can account for the known processivity of RdRp that is required for replicating the long genome of coronaviruses. Our results enable a detailed analysis of the inhibitory mechanisms that underlie the antiviral activity of substances such as remdesivir, a drug for the treatment of coronavirus disease 2019 (COVID-19).
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http://dx.doi.org/10.1038/s41586-020-2368-8DOI Listing
August 2020

Structure of H3K36-methylated nucleosome-PWWP complex reveals multivalent cross-gyre binding.

Nat Struct Mol Biol 2020 01 9;27(1):8-13. Epub 2019 Dec 9.

Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

Recognition of histone-modified nucleosomes by specific reader domains underlies the regulation of chromatin-associated processes. Whereas structural studies revealed how reader domains bind modified histone peptides, it is unclear how reader domains interact with modified nucleosomes. Here, we report the cryo-electron microscopy structure of the PWWP reader domain of human transcriptional coactivator LEDGF in complex with an H3K36-methylated nucleosome at 3.2-Å resolution. The structure reveals multivalent binding of the reader domain to the methylated histone tail and to both gyres of nucleosomal DNA, explaining the known cooperative interactions. The observed cross-gyre binding may contribute to nucleosome integrity during transcription. The structure also explains how human PWWP domain-containing proteins are recruited to H3K36-methylated regions of the genome for transcription, histone acetylation and methylation, and for DNA methylation and repair.
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http://dx.doi.org/10.1038/s41594-019-0345-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955156PMC
January 2020

Structure of transcribing RNA polymerase II-nucleosome complex.

Nat Commun 2018 12 21;9(1):5432. Epub 2018 Dec 21.

Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Am Fassberg 11, 37077, Göttingen, Germany.

Transcription of eukaryotic protein-coding genes requires passage of RNA polymerase II (Pol II) through nucleosomes, but it is unclear how this is achieved. Here we report the cryo-EM structure of transcribing Saccharomyces cerevisiae Pol II engaged with a downstream nucleosome core particle at an overall resolution of 4.4 Å. Pol II and the nucleosome are observed in a defined relative orientation that is not predicted. Pol II contacts both sides of the nucleosome dyad using its clamp head and lobe domains. Structural comparisons reveal that the elongation factors TFIIS, DSIF, NELF, SPT6, and PAF1 complex can be accommodated on the Pol II surface in the presence of the oriented nucleosome. Our results provide a starting point for analysing the mechanisms of chromatin transcription.
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http://dx.doi.org/10.1038/s41467-018-07870-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303367PMC
December 2018

The interaction landscape between transcription factors and the nucleosome.

Nature 2018 10 24;562(7725):76-81. Epub 2018 Sep 24.

Department of Biochemistry, University of Cambridge, Cambridge, UK.

Nucleosomes cover most of the genome and are thought to be displaced by transcription factors in regions that direct gene expression. However, the modes of interaction between transcription factors and nucleosomal DNA remain largely unknown. Here we systematically explore interactions between the nucleosome and 220 transcription factors representing diverse structural families. Consistent with earlier observations, we find that the majority of the studied transcription factors have less access to nucleosomal DNA than to free DNA. The motifs recovered from transcription factors bound to nucleosomal and free DNA are generally similar. However, steric hindrance and scaffolding by the nucleosome result in specific positioning and orientation of the motifs. Many transcription factors preferentially bind close to the end of nucleosomal DNA, or to periodic positions on the solvent-exposed side of the DNA. In addition, several transcription factors usually bind to nucleosomal DNA in a particular orientation. Some transcription factors specifically interact with DNA located at the dyad position at which only one DNA gyre is wound, whereas other transcription factors prefer sites spanning two DNA gyres and bind specifically to each of them. Our work reveals notable differences in the binding of transcription factors to free and nucleosomal DNA, and uncovers a diverse interaction landscape between transcription factors and the nucleosome.
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http://dx.doi.org/10.1038/s41586-018-0549-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173309PMC
October 2018

Structure of paused transcription complex Pol II-DSIF-NELF.

Nature 2018 08 22;560(7720):601-606. Epub 2018 Aug 22.

Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Göttingen, Germany.

Metazoan gene regulation often involves the pausing of RNA polymerase II (Pol II) in the promoter-proximal region. Paused Pol II is stabilized by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we report the cryo-electron microscopy structure of a paused transcription elongation complex containing Sus scrofa Pol II and Homo sapiens DSIF and NELF at 3.2 Å resolution. The structure reveals a tilted DNA-RNA hybrid that impairs binding of the nucleoside triphosphate substrate. NELF binds the polymerase funnel, bridges two mobile polymerase modules, and contacts the trigger loop, thereby restraining Pol II mobility that is required for pause release. NELF prevents binding of the anti-pausing transcription elongation factor IIS (TFIIS). Additionally, NELF possesses two flexible 'tentacles' that can contact DSIF and exiting RNA. These results define the paused state of Pol II and provide the molecular basis for understanding the function of NELF during promoter-proximal gene regulation.
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http://dx.doi.org/10.1038/s41586-018-0442-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6245578PMC
August 2018

Structure of activated transcription complex Pol II-DSIF-PAF-SPT6.

Nature 2018 08 22;560(7720):607-612. Epub 2018 Aug 22.

Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Göttingen, Germany.

Gene regulation involves activation of RNA polymerase II (Pol II) that is paused and bound by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we show that formation of an activated Pol II elongation complex in vitro requires the kinase function of the positive transcription elongation factor b (P-TEFb) and the elongation factors PAF1 complex (PAF) and SPT6. The cryo-EM structure of an activated elongation complex of Sus scrofa Pol II and Homo sapiens DSIF, PAF and SPT6 was determined at 3.1 Å resolution and compared to the structure of the paused elongation complex formed by Pol II, DSIF and NELF. PAF displaces NELF from the Pol II funnel for pause release. P-TEFb phosphorylates the Pol II linker to the C-terminal domain. SPT6 binds to the phosphorylated C-terminal-domain linker and opens the RNA clamp formed by DSIF. These results provide the molecular basis for Pol II pause release and elongation activation.
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http://dx.doi.org/10.1038/s41586-018-0440-4DOI Listing
August 2018

Cryo-EM structure of a mammalian RNA polymerase II elongation complex inhibited by α-amanitin.

J Biol Chem 2018 05 17;293(19):7189-7194. Epub 2018 Mar 17.

Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany. Electronic address:

RNA polymerase II (Pol II) is the central enzyme that transcribes eukaryotic protein-coding genes to produce mRNA. The mushroom toxin α-amanitin binds Pol II and inhibits transcription at the step of RNA chain elongation. Pol II from yeast binds α-amanitin with micromolar affinity, whereas metazoan Pol II enzymes exhibit nanomolar affinities. Here, we present the high-resolution cryo-EM structure of α-amanitin bound to and inhibited by its natural target, the mammalian Pol II elongation complex. The structure revealed that the toxin is located in a pocket previously identified in yeast Pol II but forms additional contacts with metazoan-specific residues, which explains why its affinity to mammalian Pol II is ∼3000 times higher than for yeast Pol II. Our work provides the structural basis for the inhibition of mammalian Pol II by the natural toxin α-amanitin and highlights that cryo-EM is well suited to studying interactions of a small molecule with its macromolecular target.
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http://dx.doi.org/10.1074/jbc.RA118.002545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5949985PMC
May 2018

Mechanism of RNA polymerase II stalling by DNA alkylation.

Proc Natl Acad Sci U S A 2017 11 30;114(46):12172-12177. Epub 2017 Oct 30.

Department of Health Sciences and Technology, ETH Zurich, 8092 Zurich, Switzerland;

Several anticancer agents that form DNA adducts in the minor groove interfere with DNA replication and transcription to induce apoptosis. Therapeutic resistance can occur, however, when cells are proficient in the removal of drug-induced damage. Acylfulvenes are a class of experimental anticancer agents with a unique repair profile suggesting their capacity to stall RNA polymerase (Pol) II and trigger transcription-coupled nucleotide excision repair. Here we show how different forms of DNA alkylation impair transcription by RNA Pol II in cells and with the isolated enzyme and unravel a mode of RNA Pol II stalling that is due to alkylation of DNA in the minor groove. We incorporated a model for acylfulvene adducts, the stable 3-deaza-3-methoxynaphtylethyl-adenosine analog (3d-Napht-A), and smaller 3-deaza-adenosine analogs, into DNA oligonucleotides to assess RNA Pol II transcription elongation in vitro. RNA Pol II was strongly blocked by a 3d-Napht-A analog but bypassed smaller analogs. Crystal structure analysis revealed that a DNA base containing 3d-Napht-A can occupy the +1 templating position and impair closing of the trigger loop in the Pol II active center and polymerase translocation into the next template position. These results show how RNA Pol II copes with minor-groove DNA alkylation and establishes a mechanism for drug resistance.
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http://dx.doi.org/10.1073/pnas.1706592114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5699039PMC
November 2017

Nucleosome-Chd1 structure and implications for chromatin remodelling.

Nature 2017 10 11;550(7677):539-542. Epub 2017 Oct 11.

Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Am Fassberg 11, 37077 Göttingen, Germany.

Chromatin-remodelling factors change nucleosome positioning and facilitate DNA transcription, replication, and repair. The conserved remodelling factor chromodomain-helicase-DNA binding protein 1(Chd1) can shift nucleosomes and induce regular nucleosome spacing. Chd1 is required for the passage of RNA polymerase IIthrough nucleosomes and for cellular pluripotency. Chd1 contains the DNA-binding domains SANT and SLIDE, a bilobal motor domain that hydrolyses ATP, and a regulatory double chromodomain. Here we report the cryo-electron microscopy structure of Chd1 from the yeast Saccharomyces cerevisiae bound to a nucleosome at a resolution of 4.8 Å. Chd1 detaches two turns of DNA from the histone octamer and binds between the two DNA gyres in a state poised for catalysis. The SANT and SLIDE domains contact detached DNA around superhelical location (SHL) -7 of the first DNA gyre. The ATPase motor binds the second DNA gyre at SHL +2 and is anchored to the N-terminal tail of histone H4, as seen in a recent nucleosome-Snf2 ATPase structure. Comparisons with published results reveal that the double chromodomain swings towards nucleosomal DNA at SHL +1, resulting in ATPase closure. The ATPase can then promote translocation of DNA towards the nucleosome dyad, thereby loosening the first DNA gyre and remodelling the nucleosome. Translocation may involve ratcheting of the two lobes of the ATPase, which is trapped in a pre- or post-translocation state in the absence or presence, respectively, of transition state-mimicking compounds.
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http://dx.doi.org/10.1038/nature24046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5697743PMC
October 2017

Nucleosomal arrangement affects single-molecule transcription dynamics.

Proc Natl Acad Sci U S A 2016 Nov 24;113(45):12733-12738. Epub 2016 Oct 24.

Biotechnology Center, Technical University Dresden, 01307 Dresden, Germany;

In eukaryotes, gene expression depends on chromatin organization. However, how chromatin affects the transcription dynamics of individual RNA polymerases has remained elusive. Here, we use dual trap optical tweezers to study single yeast RNA polymerase II (Pol II) molecules transcribing along a DNA template with two nucleosomes. The slowdown and the changes in pausing behavior within the nucleosomal region allow us to determine a drift coefficient, , which characterizes the ability of the enzyme to recover from a nucleosomal backtrack. Notably, can be used to predict the probability to pass the first nucleosome. Importantly, the presence of a second nucleosome changes in a manner that depends on the spacing between the two nucleosomes, as well as on their rotational arrangement on the helical DNA molecule. Our results indicate that the ability of Pol II to pass the first nucleosome is increased when the next nucleosome is turned away from the first one to face the opposite side of the DNA template. These findings help to rationalize how chromatin arrangement affects Pol II transcription dynamics.
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http://dx.doi.org/10.1073/pnas.1602764113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5111697PMC
November 2016

The structure and substrate specificity of human Cdk12/Cyclin K.

Nat Commun 2014 Mar 24;5:3505. Epub 2014 Mar 24.

1] Group Physical Biochemistry, Center of Advanced European Studies and Research, Ludwig-Erhard-Allee 2, Bonn 53175, Germany [2] Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, Dortmund 44227, Germany.

Phosphorylation of the RNA polymerase II C-terminal domain (CTD) by cyclin-dependent kinases is important for productive transcription. Here we determine the crystal structure of Cdk12/CycK and analyse its requirements for substrate recognition. Active Cdk12/CycK is arranged in an open conformation similar to that of Cdk9/CycT but different from those of cell cycle kinases. Cdk12 contains a C-terminal extension that folds onto the N- and C-terminal lobes thereby contacting the ATP ribose. The interaction is mediated by an HE motif followed by a polybasic cluster that is conserved in transcriptional CDKs. Cdk12/CycK showed the highest activity on a CTD substrate prephosphorylated at position Ser7, whereas the common Lys7 substitution was not recognized. Flavopiridol is most potent towards Cdk12 but was still 10-fold more potent towards Cdk9. T-loop phosphorylation of Cdk12 required coexpression with a Cdk-activating kinase. These results suggest the regulation of Pol II elongation by a relay of transcriptionally active CTD kinases.
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http://dx.doi.org/10.1038/ncomms4505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973122PMC
March 2014