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
• elektronová kryomikroskopie MeSH
• genetická transkripce MeSH
• histonové chaperony genetika   metabolismus   MeSH
• nukleozomy * genetika   metabolismus   MeSH
• RNA-polymerasa II metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny * metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• transkripční elongační faktory metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• histonové chaperony MeSH
• nukleozomy * MeSH
• RNA-polymerasa II MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• SPT6 protein, S cerevisiae MeSH Prohlížeč
• transkripční elongační faktory 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-dependentní DNA-polymerasy chemie   metabolismus   MeSH
• exozómy metabolismus   MeSH
• fungální RNA metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární modely MeSH
• nekódující RNA metabolismus   MeSH
• polyadenylace MeSH
• proteiny vázající RNA chemie   metabolismus   MeSH
• RNA-polymerasa II metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• stabilita RNA MeSH
• terminace genetické transkripce * MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-dependentní DNA-polymerasy MeSH
• fungální RNA MeSH
• nekódující RNA MeSH
• NRD1 protein, S cerevisiae MeSH Prohlížeč
• PAP2 protein, S cerevisiae MeSH Prohlížeč
• proteiny vázající RNA MeSH
• RNA-polymerasa II MeSH
• Saccharomyces cerevisiae - proteiny MeSH

Recruitment of appropriate RNA processing factors to the site of transcription is controlled by post-translational modifications of the C-terminal domain (CTD) of RNA polymerase II (RNAP II). Here, we report the solution structure of the Ser5 phosphorylated (pSer5) CTD bound to Nrd1. The structure reveals a direct recognition of pSer5 by Nrd1 that requires the cis conformation of the upstream pSer5-Pro6 peptidyl-prolyl bond of the CTD. Mutations at the complex interface diminish binding affinity and impair processing or degradation of noncoding RNAs. These findings underpin the interplay between covalent and noncovalent changes in the CTD structure that constitute the CTD code.

• MeSH
• fosforylace MeSH
• molekulární modely MeSH
• nekódující RNA metabolismus   MeSH
• prolin metabolismus   MeSH
• proteiny vázající RNA chemie   metabolismus   MeSH
• RNA-polymerasa II metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae cytologie   enzymologie   genetika   metabolismus   MeSH
• serin metabolismus   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• viabilita buněk MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nekódující RNA MeSH
• NRD1 protein, S cerevisiae MeSH Prohlížeč
• prolin MeSH
• proteiny vázající RNA MeSH
• RNA-polymerasa II MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• serin MeSH