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Capturing the transient, in vivo binding partners of transcriptional activators using a genetically incorporated photo‐crosslinking amino acid
Author(s) -
Dugan Amanda,
Mapp Anna K.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.550.15
Subject(s) - coactivator , activator (genetics) , transcription factor , in vivo , protein–protein interaction , microbiology and biotechnology , promoter , chemistry , gene , biology , computational biology , biochemistry , gene expression , genetics
Transcriptional activators are modular, signal responsive proteins that play an essential role in regulating gene expression. When localized to the nucleus, activators engage in a wave of interactions with a suite of multi‐protein coactivator complexes, resulting in their recruitment to the promoter of the gene to be transcribed. While the majority of methods available for studying protein‐protein interactions (PPIs) are ideal for studying those of higher affinity, few are well‐suited for studying PPIs of a more moderate affinity, including activator‐coactivator interactions. Recently, covalent crosslinking using a photoactivatable amino acid (Bpa) has proven to be a powerful method for the in vivo capture of moderate affinity PPIs. Herein we use in vivo photocrosslinking to examine the interaction between the amphipathic activator VP16 and the essential transcription factor TBP in live yeast. Several lines of evidence suggest that TBP is recruited to promoters through a direct interaction with VP16; However, in vivo co‐localization studies suggest that this recruitment occurs through an indirect mechanism. Using this methodology, we find that VP16 does in fact directly interact with TBP and that this interaction is sustained on DNA. We also find that TBP is a shared target of other amphipathic activators, suggesting a common mechanism of recruitment within a single family of activator proteins. Thus, this methodology should be useful in studying regulatory proteins involved in complex interaction networks. This project is funded by NIH‐NIGMS 2R0106553.