A tethered catalysis, two-hybrid system to identify protein-protein interactions requiring post-translational modifications.
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.
Guo
Dawei
D
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.
Hazbun
Tony R
TR
Xu
Xin-Jing
XJ
Ng
Sze-Ling
SL
Fields
Stanley
S
Kuo
Min-Hao
MH
D000107:Acetylation; D002384:Catalysis; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D004268:DNA-Binding Proteins; D006657:Histones; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; D012319:RNA Polymerase II