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

The transcription factor ASCIZ (ATMIN, ZNF822) has an unusually high number of recognition motifs for the product of its main target gene, the hub protein LC8 (DYNLL1). Using a combination of biophysical methods, structural analysis by NMR and electron microscopy, and cellular transcription assays, we developed a model that proposes a concerted role of intrinsic disorder and multiple LC8 binding events in regulating LC8 transcription. We demonstrate that the long intrinsically disordered C-terminal domain of ASCIZ binds LC8 to form a dynamic ensemble of complexes with a gradient of transcriptional activity that is inversely proportional to LC8 occupancy. The preference for low occupancy complexes at saturating LC8 concentrations with both human and Drosophila ASCIZ indicates that negative cooperativity is an important feature of ASCIZ-LC8 interactions. The prevalence of intrinsic disorder and multivalency among transcription factors suggests that formation of heterogeneous, dynamic complexes is a widespread mechanism for tuning transcriptional regulation.

• Keywords
• Intrinsically disordered proteins, NMR, dynamic complexes, electron microscopy, human, molecular biophysics, multivalency, structural biology, transcription factors,
• MeSH
• Cytoplasmic Dyneins chemistry   genetics   metabolism   MeSH
• Drosophila melanogaster growth & development   metabolism   physiology   MeSH
• Dyneins chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Drosophila Proteins chemistry   genetics   metabolism   MeSH
• Gene Expression Regulation * MeSH
• Transcription Factors chemistry   genetics   metabolism   MeSH
• Intrinsically Disordered Proteins genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• ATMIN protein, human MeSH Browser
• ctp protein, Drosophila MeSH Browser
• Cytoplasmic Dyneins MeSH
• Dyneins MeSH
• DYNLL1 protein, human MeSH Browser
• Drosophila Proteins MeSH
• Transcription Factors MeSH
• Intrinsically Disordered Proteins 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 Eighth Central European Conference "Chemistry towards Biology" was held in Brno, Czech Republic, on August 28-September 1, 2016 to bring together experts in biology, chemistry and design of bioactive compounds; promote the exchange of scientific results, methods and ideas; and encourage cooperation between researchers from all over the world. The topics of the conference covered "Chemistry towards Biology", meaning that the event welcomed chemists working on biology-related problems, biologists using chemical methods, and students and other researchers of the respective areas that fall within the common scope of chemistry and biology. The authors of this manuscript are plenary speakers and other participants of the symposium and members of their research teams. The following summary highlights the major points/topics of the meeting.

• Keywords
• ADME, drug delivery systems, biological chemistry, biomaterials, chemical biology, drug design, nanoparticles, natural compounds, proteins and nucleic acids, synthesis, targeting,
• MeSH
• Epigenesis, Genetic MeSH
• Chemistry, Pharmaceutical methods   MeSH
• Drug Delivery Systems MeSH
• Proteins chemistry   MeSH
• Drug Design MeSH
• Systems Biology MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Congress MeSH
• Names of Substances
• Proteins MeSH

Microtubule-associated proteins (MAPs) are abundantly present in axons and dendrites, and have been shown to play crucial role during the neuronal morphogenesis. The period of main dendritic outgrowth and synaptogenesis coincides with high expression levels of one of MAPs, the MAP2c, in rats. The MAP2c is a 49.2 kDa intrinsically disordered protein. To achieve an atomic resolution characterization of such a large protein, we have developed a protocol based on the acquisition of two five-dimensional (13)C-directly detected NMR experiments. Our previously published 5D CACONCACO experiment (Nováček et al. in J Biomol NMR 50(1):1-11, 2011) provides the sequential assignment of the backbone resonances, which is not interrupted by the presence of the proline residues in the amino acid sequence. A novel 5D HC(CC-TOCSY)CACON experiment facilitates the assignment of the aliphatic side chain resonances. To streamline the data analysis, we have developed a semi-automated procedure for signal assignments. The obtained data provides the first atomic resolution insight into the conformational state of MAP2c and constitutes a model for further functional studies of MAPs.

• MeSH
• Algorithms MeSH
• Glycine MeSH
• Rats MeSH
• Molecular Sequence Data MeSH
• Molecular Weight MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Microtubule-Associated Proteins chemistry   metabolism   MeSH
• Protein Structure, Secondary MeSH
• Amino Acid Sequence MeSH
• Intrinsically Disordered Proteins chemistry   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glycine MeSH
• Microtubule-Associated Proteins MeSH
• Intrinsically Disordered Proteins MeSH

A pair of 4D NMR experiments for the backbone assignment of disordered proteins is presented. The experiments exploit (13)C direct detection and non-uniform sampling of the indirectly detected dimensions, and provide correlations of the aliphatic proton (H(α), and H(β)) and carbon (C(α), C(β)) resonance frequencies to the protein backbone. Thus, all the chemical shifts regularly used to map the transient secondary structure motifs in the intrinsically disordered proteins (H(α), C(α), C(β), C', and N) can be extracted from each spectrum. Compared to the commonly used assignment strategy based on matching the C(α) and C(β) chemical shifts, inclusion of the H(α) and H(β) provides up to three extra resonance frequencies that decrease the chance of ambiguous assignment. The experiments were successfully applied to the original assignment of a 12.8 kDa intrinsically disordered protein having a high content of proline residues (26 %) in the sequence.

• MeSH
• Carbon Isotopes chemistry   MeSH
• Molecular Sequence Data MeSH
• Nuclear Magnetic Resonance, Biomolecular methods   MeSH
• Proline chemistry   MeSH
• Proteins chemistry   MeSH
• Protein Structure, Secondary MeSH
• Amino Acid Sequence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Carbon Isotopes MeSH
• Proline MeSH
• Proteins MeSH