Nature Biotechnology22, 888 - 892 (2004)
Published online: 20 June 2004; | doi:10.1038/nbt985
A tethered catalysis, two-hybrid system to identify protein-protein interactions requiring post-translational modifications
Dawei Guo1, 4, Tony R Hazbun2, 4, Xin-Jing Xu1, Sze-Ling Ng1, Stanley Fields2
& Min-Hao Kuo1, 3
1
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.
2
Howard Hughes Medical Institute, Departments of Genome Sciences and Medicine, University of Washington, Box 357730, Seattle, Washington 98195, USA.
3
Programs in Cell and Molecular Biology and Genetics, Michigan State University, East Lansing, Michigan 48824, USA.
4
These authors contributed equally to this work.
Correspondence should be addressed to Min-Hao Kuo kuom@msu.edu
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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