Development and Implementation of In Vivo Crosslinking for Identifying Protein Targets of the Transcriptional Activator Gal4.

[摘要] Protein-protein interactions are essential to biological processes; therefore, developing methods to discover and characterize these interactions is of high importance to the scientific community.Transcription, a fundamental cellular function, relies upon protein-protein interactions between a transcriptional activator and the protein complexes that are recruited to DNA in order to initiate the process. Given the relationship between transcription misregulation and disease, protein-protein interactions responsible for transcriptional regulation are attractive targets for therapeutic intervention. Despite considerable effort, the discovery of the direct and functionally relevant protein binding partners of transcriptional activation domains (TADs) of the amphipathic activator class has proven to be challenging, with a number of conflicting models. To create a detailed network map filling this void, an enhanced tRNA/tRNA synthetase pair has been developed to site specifically incorporate the photolabile nonnatural amino acid p-benzoyl-L-phenylalanine (pBpa) in vivo (S. cerevisiae) into the amphipathic prototypical transcriptional activator Gal4 to capture the interactions through in vivo photo-crosslinking. Applying this methodology, in vivo photo-crosslinking was used to detect a key binding partner of Gal4, the inhibitor protein Gal80.This approach when followed by mass spectrometric analysis of crosslinked protein partners also resulted in identifying Gal80 as a protein in the purified crosslinked reaction mixture.Using this technique other potential targets of Gal4 were identified under conditions where Gal80 is not inhibiting Gal4-activated transcription.Some of these potential targets, Ccr4, Mot2, Tra1, Ssl2, Mlp2, Abf1, Ctr9, Swi1, Hrr25, are consistent with previously suggested targets of amphipathic transcriptional activators while others are novel findings.Further verification of these targets will be will be critical for elucidating the mechanism of transcriptional activation, useful information for developing therapeutics designed to regulate aberrant gene expression in diseases.

[发布机构] University of Michigan

[效力级别] Biological Chemistry

[关键词] In Vivo Crosslinking of the Transcriptional Activator Gal4;Biological Chemistry;Science;Chemical Biology