The intrinsically disordered carboxy-terminal domain (CTD) of the largest subunit of RNA Polymerase II (RNAPII) consists of multiple tandem repeats of the consensus heptapeptide Y1-S2-P3-T4-S5-P6-S7. The CTD promotes liquid-liquid phase-separation (LLPS) of RNAPII in vivo. However, understanding the role of the conserved heptad residues in LLPS is hampered by the lack of direct biochemical characterization of the CTD. Here, we generated a systematic array of CTD variants to unravel the sequence-encoded molecular grammar underlying the LLPS of the human CTD. Using in vitro experiments and molecular dynamics simulations, we report that the aromaticity of tyrosine and cis-trans isomerization of prolines govern CTD phase-separation. The cis conformation of prolines and β-turns in the SPXX motif contribute to a more compact CTD ensemble, enhancing interactions among CTD residues. We further demonstrate that prolines and tyrosine in the CTD consensus sequence are required for phosphorylation by Cyclin-dependent kinase 7 (CDK7). Under phase-separation conditions, CDK7 associates with the surface of the CTD droplets, drastically accelerating phosphorylation and promoting the release of hyperphosphorylated CTD from the droplets. Our results highlight the importance of conformationally restricted local structures within spacer regions, separating uniformly spaced tyrosine stickers of the CTD heptads, which are required for CTD phase-separation.

• MeSH
• Cyclin-Dependent Kinases * metabolism   chemistry   MeSH
• Phosphorylation MeSH
• Cyclin-Dependent Kinase-Activating Kinase * MeSH
• Humans MeSH
• Proline metabolism   chemistry   MeSH
• Protein Domains MeSH
• RNA Polymerase II * metabolism   chemistry   MeSH
• Amino Acid Sequence MeSH
• Molecular Dynamics Simulation * MeSH
• Tyrosine metabolism   chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CDK7 protein, human MeSH Browser
• Cyclin-Dependent Kinases * MeSH
• Cyclin-Dependent Kinase-Activating Kinase * MeSH
• Proline MeSH
• RNA Polymerase II * MeSH
• Tyrosine MeSH

Transcription elongation factor Spt6 associates with RNA polymerase II (Pol II) and acts as a histone chaperone, which promotes the reassembly of nucleosomes following the passage of Pol II. The precise mechanism of nucleosome reassembly mediated by Spt6 remains unclear. In this study, we used a hybrid approach combining cryo-electron microscopy and small-angle X-ray scattering to visualize the architecture of Spt6 from Saccharomyces cerevisiae. The reconstructed overall architecture of Spt6 reveals not only the core of Spt6, but also its flexible N- and C-termini, which are critical for Spt6's function. We found that the acidic N-terminal region of Spt6 prevents the binding of Spt6 not only to the Pol II CTD and Pol II CTD-linker, but also to pre-formed intact nucleosomes and nucleosomal DNA. The N-terminal region of Spt6 self-associates with the tSH2 domain and the core of Spt6 and thus controls binding to Pol II and nucleosomes. Furthermore, we found that Spt6 promotes the assembly of nucleosomes in vitro. These data indicate that the cooperation between the intrinsically disordered and structured regions of Spt6 regulates nucleosome and Pol II CTD binding, and also nucleosome assembly.

• MeSH
• Cryoelectron Microscopy MeSH
• Transcription, Genetic MeSH
• Histone Chaperones genetics   metabolism   MeSH
• Nucleosomes * genetics   metabolism   MeSH
• RNA Polymerase II metabolism   MeSH
• Saccharomyces cerevisiae Proteins * metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Transcriptional Elongation Factors metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Histone Chaperones MeSH
• Nucleosomes * MeSH
• RNA Polymerase II MeSH
• Saccharomyces cerevisiae Proteins * MeSH
• SPT6 protein, S cerevisiae MeSH Browser
• Transcriptional Elongation Factors MeSH

Transcription elongation factor Spt6 associates with RNA polymerase II (RNAP II) via a tandem SH2 (tSH2) domain. The mechanism and significance of the RNAP II-Spt6 interaction is still unclear. Recently, it was proposed that Spt6-tSH2 is recruited via a newly described phosphorylated linker between the Rpb1 core and its C-terminal domain (CTD). Here, we report binding studies with isolated tSH2 of Spt6 (Spt6-tSH2) and Spt6 lacking the first unstructured 297 residues (Spt6ΔN) with a minimal CTD substrate of two repetitive heptads phosphorylated at different sites. The data demonstrate that Spt6 also binds the phosphorylated CTD, a site that was originally proposed as a recognition epitope. We also show that an extended CTD substrate harboring 13 repetitive heptads of the tyrosine-phosphorylated CTD binds Spt6-tSH2 and Spt6ΔN with tighter affinity than the minimal CTD substrate. The enhanced binding is achieved by avidity originating from multiple phosphorylation marks present in the CTD. Interestingly, we found that the steric effects of additional domains in the Spt6ΔN construct partially obscure the binding of the tSH2 domain to the multivalent ligand. We show that Spt6-tSH2 binds various phosphorylation patterns in the CTD and found that the studied combinations of phospho-CTD marks (1,2; 1,5; 2,4; and 2,7) all facilitate the interaction of CTD with Spt6. Our structural studies reveal a plasticity of the tSH2 binding pockets that enables the accommodation of CTDs with phosphorylation marks in different registers.

• Keywords
• CTD, RNA polymerase II, Spt6, NMR structure, phosphorylation,
• MeSH
• Epitopes genetics   MeSH
• Phosphorylation genetics   MeSH
• Transcription, Genetic * MeSH
• Histone Chaperones genetics   MeSH
• RNA Polymerase II genetics   MeSH
• Saccharomyces cerevisiae Proteins genetics   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Amino Acid Sequence genetics   MeSH
• src Homology Domains genetics   MeSH
• Transcriptional Elongation Factors genetics   MeSH
• Protein Binding genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Epitopes MeSH
• Histone Chaperones MeSH
• RNA Polymerase II MeSH
• Saccharomyces cerevisiae Proteins MeSH
• SPT6 protein, S cerevisiae MeSH Browser
• Transcriptional Elongation Factors MeSH

Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non-coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a threat to proper gene expression that needs to be controlled. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non-coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here, we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II, and structurally characterize its recognition by the CTD-interacting domain of Nrd1, an RNA-binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1-dependent termination strictly requires CTD recognition by the N-terminal domain of Sen1. We provide evidence that the Sen1-CTD interaction does not promote initial Sen1 recruitment, but rather enhances Sen1 capacity to induce the release of paused RNAPII from the DNA. Our results shed light on the network of protein-protein interactions that control termination of non-coding transcription by Sen1.

• Keywords
• RNA polymerase II CTD, Sen1 helicase, non-coding transcription, pervasive transcription, transcription termination,
• MeSH
• DNA Helicases chemistry   metabolism   MeSH
• RNA, Fungal metabolism   MeSH
• Protein Conformation MeSH
• Models, Molecular MeSH
• RNA, Untranslated metabolism   MeSH
• Protein Domains MeSH
• RNA-Binding Proteins chemistry   metabolism   MeSH
• Gene Expression Regulation, Fungal MeSH
• RNA Helicases chemistry   metabolism   MeSH
• RNA Polymerase II chemistry   MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Transcription Termination, Genetic MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Helicases MeSH
• RNA, Fungal MeSH
• RNA, Untranslated MeSH
• NRD1 protein, S cerevisiae MeSH Browser
• RNA-Binding Proteins MeSH
• RNA Helicases MeSH
• RNA Polymerase II MeSH
• Saccharomyces cerevisiae Proteins MeSH
• SEN1 protein, S cerevisiae MeSH Browser

RNA polymerase II contains a long C-terminal domain (CTD) that regulates interactions at the site of transcription. The CTD architecture remains poorly understood due to its low sequence complexity, dynamic phosphorylation patterns, and structural variability. We used integrative structural biology to visualize the architecture of the CTD in complex with Rtt103, a 3'-end RNA-processing and transcription termination factor. Rtt103 forms homodimers via its long coiled-coil domain and associates densely on the repetitive sequence of the phosphorylated CTD via its N-terminal CTD-interacting domain. The CTD-Rtt103 association opens the compact random coil structure of the CTD, leading to a beads-on-a-string topology in which the long rod-shaped Rtt103 dimers define the topological and mobility restraints of the entire assembly. These findings underpin the importance of the structural plasticity of the CTD, which is templated by a particular set of CTD-binding proteins.

• Keywords
• CTD, RNA polymerase II, Rtt103, structural biology, transcription,
• MeSH
• Protein Interaction Domains and Motifs MeSH
• Crystallography, X-Ray MeSH
• Magnetic Resonance Spectroscopy MeSH
• Protein Multimerization MeSH
• RNA Polymerase II metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Transcription Factors chemistry   metabolism   MeSH
• Publication type
• Video-Audio Media MeSH
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA Polymerase II MeSH
• Rtt103 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• Transcription Factors MeSH

Phosphorylation patterns of the C-terminal domain (CTD) of largest subunit of RNA polymerase II (called the CTD code) orchestrate the recruitment of RNA processing and transcription factors. Recent studies showed that not only serines and tyrosines but also threonines of the CTD can be phosphorylated with a number of functional consequences, including the interaction with yeast transcription termination factor, Rtt103p. Here, we report the solution structure of the Rtt103p CTD-interacting domain (CID) bound to Thr4 phosphorylated CTD, a poorly understood letter of the CTD code. The structure reveals a direct recognition of the phospho-Thr4 mark by Rtt103p CID and extensive interactions involving residues from three repeats of the CTD heptad. Intriguingly, Rtt103p's CID binds equally well Thr4 and Ser2 phosphorylated CTD A doubly phosphorylated CTD at Ser2 and Thr4 diminishes its binding affinity due to electrostatic repulsion. Our structural data suggest that the recruitment of a CID-containing CTD-binding factor may be coded by more than one letter of the CTD code.

• Keywords
• NMR, RNA processing, RNAPII CTD code, structural biology,
• MeSH
• Phosphorylation MeSH
• Transcription, Genetic MeSH
• Protein Kinases metabolism   MeSH
• Proteolysis MeSH
• RNA Polymerase II chemistry   metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Serine metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Threonine chemistry   metabolism   MeSH
• Transcription Factors chemistry   metabolism   MeSH
• Tyrosine metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Protein Kinases MeSH
• RNA Polymerase II MeSH
• Rtt103 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• Serine MeSH
• Threonine MeSH
• Transcription Factors MeSH
• Tyrosine MeSH

The Nrd1-Nab3-Sen1 (NNS) complex is essential for controlling pervasive transcription and generating sn/snoRNAs in S. cerevisiae. The NNS complex terminates transcription of noncoding RNA genes and promotes exosome-dependent processing/degradation of the released transcripts. The Trf4-Air2-Mtr4 (TRAMP) complex polyadenylates NNS target RNAs and favors their degradation. NNS-dependent termination and degradation are coupled, but the mechanism underlying this coupling remains enigmatic. Here we provide structural and functional evidence demonstrating that the same domain of Nrd1p interacts with RNA polymerase II and Trf4p in a mutually exclusive manner, thus defining two alternative forms of the NNS complex, one involved in termination and the other in degradation. We show that the Nrd1-Trf4 interaction is required for optimal exosome activity in vivo and for the stimulation of polyadenylation of NNS targets by TRAMP in vitro. We propose that transcription termination and RNA degradation are coordinated by switching between two alternative partners of the NNS complex.

• MeSH
• DNA-Directed DNA Polymerase chemistry   metabolism   MeSH
• Exosomes metabolism   MeSH
• RNA, Fungal metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Magnetic Resonance Spectroscopy MeSH
• Models, Molecular MeSH
• RNA, Untranslated metabolism   MeSH
• Polyadenylation MeSH
• RNA-Binding Proteins chemistry   metabolism   MeSH
• RNA Polymerase II metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• RNA Stability MeSH
• Transcription Termination, Genetic * MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Directed DNA Polymerase MeSH
• RNA, Fungal MeSH
• RNA, Untranslated MeSH
• NRD1 protein, S cerevisiae MeSH Browser
• PAP2 protein, S cerevisiae MeSH Browser
• RNA-Binding Proteins MeSH
• RNA Polymerase II MeSH
• Saccharomyces cerevisiae Proteins MeSH

In Saccharomyces cerevisiae, the Nrd1-dependent termination and processing pathways play an important role in surveillance and processing of non-coding ribonucleic acids (RNAs). The termination and subsequent processing is dependent on the Nrd1 complex consisting of two RNA-binding proteins Nrd1 and Nab3 and Sen1 helicase. It is established that Nrd1 and Nab3 cooperatively recognize specific termination elements within nascent RNA, GUA[A/G] and UCUU[G], respectively. Interestingly, some transcripts do not require GUA[A/G] motif for transcription termination in vivo and binding in vitro, suggesting the existence of alternative Nrd1-binding motifs. Here we studied the structure and RNA-binding properties of Nrd1 using nuclear magnetic resonance (NMR), fluorescence anisotropy and phenotypic analyses in vivo. We determined the solution structure of a two-domain RNA-binding fragment of Nrd1, formed by an RNA-recognition motif and helix-loop bundle. NMR and fluorescence data show that not only GUA[A/G] but also several other G-rich and AU-rich motifs are able to bind Nrd1 with affinity in a low micromolar range. The broad substrate specificity is achieved by adaptable interaction surfaces of the RNA-recognition motif and helix-loop bundle domains that sandwich the RNA substrates. Our findings have implication for the role of Nrd1 in termination and processing of many non-coding RNAs arising from bidirectional pervasive transcription.

• MeSH
• Dimerization MeSH
• Models, Molecular MeSH
• Mutation MeSH
• RNA-Binding Proteins chemistry   genetics   metabolism   MeSH
• RNA chemistry   metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• NRD1 protein, S cerevisiae MeSH Browser
• RNA-Binding Proteins MeSH
• RNA MeSH
• Saccharomyces cerevisiae Proteins MeSH