Abstract
Phosphorylation of the C-terminal domain (CTD) of RNA polymerase II controls the co-transcriptional assembly of RNA processing and transcription factors. Recruitment relies on conserved CTD-interacting domains (CIDs) that recognize different CTD phosphoisoforms during the transcription cycle, but the molecular basis for their specificity remains unclear. We show that the CIDs of two transcription termination factors, Rtt103 and Pcf11, achieve high affinity and specificity both by specifically recognizing the phosphorylated CTD and by cooperatively binding to neighboring CTD repeats. Single-residue mutations at the protein-protein interface abolish cooperativity and affect recruitment at the 3′ end processing site in vivo. We suggest that this cooperativity provides a signal-response mechanism to ensure that its action is confined only to proper polyadenylation sites where Ser2 phosphorylation density is highest.
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Acknowledgements
We thank R. Becker for his assistance with the fluorescence anisotropy experiments. We also thank B. Demeler and V. Schirf at the Center for Analytical Ultracentrifugation of Macromolecular Assemblies for assistance with the AUC experiments. This work was supported by grants from the National Institute of General Medical Sciences of the US National Institutes of Health to G.V. (GM64440) and S.B. (GM56663) and from the German Research Foundation to A.M. (ME3135/1-1) as well as by the WCU project (305-20080089) from KMEST and grants from the National Research Foundation of Korea (NRF) (R31-2009-000-10032-0 and 2010-0011750) to M.K. A portion of the research was performed using the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research located at Pacific Northwest National Laboratory.
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B.M.L. carried out all NMR titration and fluorescence anisotropy experiments with the four-heptad repeat CTD peptides. Diheptad repeat fluorescence anisotropy experiments were done by H.M. H.M. wrote the scripts for fitting the fluorescence anisotropy measurements. B.M.L. and H.M. analyzed the fluorescence anisotropy data. Structure determination of Rtt103-CID was done by S.L.R. Rtt103-CID bound to the Ser2P CTD was determined by B.M.L. Isotope-filtered NMR experiments were collected by T.C.L. All mutants were made by B.M.L., while F.Y. collected and analyzed NMR relaxation experiments. A.M. made the Pcf11-CID and Rtt103-CID constructs and developed the expression and purification conditions. S.B., M.K. and H.S. designed the in vivo ChIP experiments, and M.K. and H.S. constructed the strains and carried out the assays. B.M.L., S.L.R., H.S., S.B., A.M. and G.V. wrote the paper.
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Lunde, B., Reichow, S., Kim, M. et al. Cooperative interaction of transcription termination factors with the RNA polymerase II C-terminal domain. Nat Struct Mol Biol 17, 1195–1201 (2010). https://doi.org/10.1038/nsmb.1893
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DOI: https://doi.org/10.1038/nsmb.1893
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