title: Detecting phosphorylation-dependent interactions with the C-terminal domain of RNA polymerase II subunit Rpb1p using a yeast two-hybrid assay.
abstract: Rpb1p, the largest subunit of S. cerevisiae RNA polymerase II, contains a repetitive structure called the C-terminal domain (CTD). The CTD serves as a scaffold for the regulated association and dissociation of more than a hundred proteins involved in RNA synthesis. Phosphorylation of two serine residues (Ser(2) and Ser(5)) in the repeating units of the CTD change dynamically during the pre-initiation, initiation, elongation and termination of transcription to control the binding and release of transcriptional components. A modification of the well established yeast two-hybrid assay for protein-protein interactions is described that detects interactions between phosphorylated forms of the CTD and proteins whose interactions with the CTD depend on phosphorylation. The efficacy of the approach was established by first showing that two-hybrid fusions containing the CTD are phosphorylated at Ser(2) and Ser(5) residues. Interactions between the CTD and three known CTD-binding proteins were analyzed. The results suggest that the modified two-hybrid system accurately assays CTD-binding and provides a new and convenient assay for CTD-binding proteins.
authors.affiliation: Laboratories of Genetics and Molecular biology, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Doris
authors.initials: D
authors.lastname: Ursic
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Finkel
authors.forename: Michael R
authors.initials: MR
authors.lastname: Culbertson
mesh_terms: D020816:Amino Acid Motifs; D010766:Phosphorylation; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine; D012319:RNA Polymerase II; C511736:RPB1 protein, S cerevisiae