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