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.

• Klíčová slova
• CTD, RNA polymerase II, Spt6, NMR structure, phosphorylation,
• MeSH
• epitopy genetika   MeSH
• fosforylace genetika   MeSH
• genetická transkripce * MeSH
• histonové chaperony genetika   MeSH
• RNA-polymerasa II genetika   MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• sekvence aminokyselin genetika   MeSH
• src homologní domény genetika   MeSH
• transkripční elongační faktory genetika   MeSH
• vazba proteinů genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• epitopy MeSH
• histonové chaperony MeSH
• RNA-polymerasa II MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• SPT6 protein, S cerevisiae MeSH Prohlížeč
• transkripční elongační faktory 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.

• Klíčová slova
• RNA polymerase II CTD, Sen1 helicase, non-coding transcription, pervasive transcription, transcription termination,
• MeSH
• DNA-helikasy chemie   metabolismus   MeSH
• fungální RNA metabolismus   MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• nekódující RNA metabolismus   MeSH
• proteinové domény MeSH
• proteiny vázající RNA chemie   metabolismus   MeSH
• regulace genové exprese u hub MeSH
• RNA-helikasy chemie   metabolismus   MeSH
• RNA-polymerasa II chemie   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• terminace genetické transkripce MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-helikasy MeSH
• fungální RNA MeSH
• nekódující RNA MeSH
• NRD1 protein, S cerevisiae MeSH Prohlížeč
• proteiny vázající RNA MeSH
• RNA-helikasy MeSH
• RNA-polymerasa II MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• SEN1 protein, S cerevisiae MeSH Prohlížeč

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.

• Klíčová slova
• Intrinsically disordered proteins, NMR, dynamic complexes, electron microscopy, human, molecular biophysics, multivalency, structural biology, transcription factors,
• MeSH
• cytoplazmatické dyneiny chemie   genetika   metabolismus   MeSH
• Drosophila melanogaster růst a vývoj   metabolismus   fyziologie   MeSH
• dyneiny chemie   genetika   metabolismus   MeSH
• lidé MeSH
• proteiny Drosophily chemie   genetika   metabolismus   MeSH
• regulace genové exprese * MeSH
• transkripční faktory chemie   genetika   metabolismus   MeSH
• vnitřně neuspořádané proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• ATMIN protein, human MeSH Prohlížeč
• ctp protein, Drosophila MeSH Prohlížeč
• cytoplazmatické dyneiny MeSH
• dyneiny MeSH
• DYNLL1 protein, human MeSH Prohlížeč
• proteiny Drosophily MeSH
• transkripční faktory MeSH
• vnitřně neuspořádané proteiny 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.

• Klíčová slova
• NMR, RNA processing, RNAPII CTD code, structural biology,
• MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• proteinkinasy metabolismus   MeSH
• proteolýza MeSH
• RNA-polymerasa II chemie   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• serin metabolismus   MeSH
• terciární struktura proteinů MeSH
• threonin chemie   metabolismus   MeSH
• transkripční faktory chemie   metabolismus   MeSH
• tyrosin metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteinkinasy MeSH
• RNA-polymerasa II MeSH
• Rtt103 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• serin MeSH
• threonin MeSH
• transkripční faktory MeSH
• tyrosin 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.

• Klíčová slova
• ADME, drug delivery systems, biological chemistry, biomaterials, chemical biology, drug design, nanoparticles, natural compounds, proteins and nucleic acids, synthesis, targeting,
• MeSH
• epigeneze genetická MeSH
• farmaceutická chemie metody   MeSH
• lékové transportní systémy MeSH
• proteiny chemie   MeSH
• racionální návrh léčiv MeSH
• systémová biologie MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• kongresy MeSH
• Názvy látek
• proteiny 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
• algoritmy MeSH
• glycin MeSH
• krysa rodu Rattus MeSH
• molekulární sekvence - údaje MeSH
• molekulová hmotnost MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• proteiny asociované s mikrotubuly chemie   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• vnitřně neuspořádané proteiny chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glycin MeSH
• proteiny asociované s mikrotubuly MeSH
• vnitřně neuspořádané proteiny 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
• izotopy uhlíku chemie   MeSH
• molekulární sekvence - údaje MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• prolin chemie   MeSH
• proteiny chemie   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• izotopy uhlíku MeSH
• prolin MeSH
• proteiny MeSH