Yeast Spt6 Reads Multiple Phosphorylation Patterns of RNA Polymerase II C-Terminal Domain In Vitro
| Autor: | Magdaléna Krejčíková, Pavel Brázda, Eliška Šmiřáková, Karel Kubíček, Aiste Kasiliauskaite, Robert Vácha, Richard Štefl, Tomáš Klumpler |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Saccharomyces cerevisiae Proteins
Transcription Genetic viruses genetic processes MST microscale thermophoresis RNA polymerase II Saccharomyces cerevisiae environment and public health Epitope Article Spt6 NMR structure src Homology Domains 03 medical and health sciences Epitopes 0302 clinical medicine Structural Biology NOESY nuclear Overhauser enhancement spectroscopy Histone Chaperones tSH2 tandem SH2 Amino Acid Sequence Molecular Biology 030304 developmental biology 0303 health sciences biology Chemistry phosphorylation C-terminus FA fluorescence anisotropy CID CTD-interacting domain HSQC heteronuclear single quantum coherence Ligand (biochemistry) CTD CTD C-terminal domain 3. Good health Cell biology A-site enzymes and coenzymes (carbohydrates) biology.protein health occupations Phosphorylation Transcriptional Elongation Factors RNAP II RNA polymerase II Linker 030217 neurology & neurosurgery NIM Nrd1 Interaction Motif Protein Binding |
| Zdroj: | Journal of Molecular Biology |
| ISSN: | 1089-8638 |
| Popis: | 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. Graphical abstract Unlabelled Image Highlights • High-affinity Pol II CTD-binding by Spt6 is achieved by avidity originating from multiple phosphorylation marks presented in the CTD, suggesting how phosphorylation levels fine-tune the CTD interactome. • Structure of RNAP II CTD bound with tandem SH2 domain of Spt6 reveals how phosphorylated CTD is recognized. • Isolated tSH2 of Spt6 binds the extended CTD substrate with tighter affinity than nearly full-length Spt6, suggesting that the steric effects of additional domains in Spt6 influence the binding of the tSH2 domain to the multivalent CTD ligand. |
| Databáze: | OpenAIRE |
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