Yeast Spt6 Reads Multiple Phosphorylation Patterns of RNA Polymerase II C-Terminal Domain In Vitro

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Authors

BRÁZDA Pavel KREJČÍKOVÁ Magdaléna KASILIAUSKAITE Aiste ŠMIŘÁKOVÁ Eliška KLUMPLER Tomáš VÁCHA Robert KUBÍČEK Karel ŠTEFL Richard

Year of publication 2020
Type Article in Periodical
Magazine / Source Journal of Molecular Biology
MU Faculty or unit

Central European Institute of Technology

Citation
Web https://www.sciencedirect.com/science/article/pii/S002228362030348X?via%3Dihub
Doi http://dx.doi.org/10.1016/j.jmb.2020.05.007
Keywords CTD; RNA polymerase II; Spt6; NMR structure; phosphorylation
Description 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 (Spt6N) 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 Spt6N 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 Spt6N 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.
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