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A tethered catalysis, two-hybrid system to identify protein-protein interactions requiring post-translational modifications

Abstract

We have modified the yeast two-hybrid system to enable the detection of protein-protein interactions that require a specific post-translational modification, using the acetylation of histones and the phosphorylation of the carboxyl terminal domain (CTD) of RNA polymerase II as test modifications. In this tethered catalysis assay, constitutive modification of the protein to be screened for interactions is achieved by fusing it to its cognate modifying enzyme, with the physical linkage resulting in efficient catalysis. This catalysis maintains substrate modification even in the presence of antagonizing enzyme activities. A catalytically inactive mutant of the enzyme is fused to the substrate as a control such that the modification does not occur; this construct enables the rapid identification of modification-independent interactions. We identified proteins with links to chromatin functions that interact with acetylated histones, and proteins that participate in RNA polymerase II functions and in CTD phosphorylation regulation that interact preferentially with the phosphorylated CTD.

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Figure 1: Acetylation of H3 and H4 when tethered to the catalytic domain of wild-type Gcn5.
Figure 2: Rpm2 and Rmt1 bind acetylated histones.
Figure 3: Autophosphorylation of CTD-Kin28 chimera.

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Acknowledgements

We thank David Arnosti and Steven Triezenberg for critical reading of this manuscript, Steven Triezenberg for suggesting the name of 'tethered catalysis,' Asha Acharya for chicken histones and bacterial lysate preparation, Kanchan Champhekar for CTD-Kin28 plasmid construction and members of the Triezenberg and Kuo labs for helpful discussion throughout this work. This work was supported by National Institutes of Health grant R01 GM62282 (M.-H.K.) and P41 RR11823 (S.F.). S.F. is an investigator of the Howard Hughes Medical Institute.

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Correspondence to Min-Hao Kuo.

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Supplementary information

Supplementary Table 1

Summary of acetylated histone-binding proteins identified by the array approach. (PDF 180 kb)

Supplementary Table 2

Summary of phosphorylated CTD-binding proteins identified by the array approach. (PDF 104 kb)

Supplementary Figure 1

Growth tests of various yeast ORFs for their abilities to interact with CTD or Kin28. (PDF 501 kb)

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Guo, D., Hazbun, T., Xu, XJ. et al. A tethered catalysis, two-hybrid system to identify protein-protein interactions requiring post-translational modifications. Nat Biotechnol 22, 888–892 (2004). https://doi.org/10.1038/nbt985

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