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Identification and Characterization of Protein‐Protein Interactions between Yeast Rap1p and the TFIID Complex
Author(s) -
Layer Justin Harrison,
Weil Peter Anthony
Publication year - 2008
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.22.1_supplement.782.20
Subject(s) - taf4 , taf1 , biology , transcription factor ii d , genetics , taf2 , microbiology and biotechnology , transcription factor , transcription factor ii a , general transcription factor , gene , promoter , gene expression , enhancer
In yeast transcription of Ribosomal Protein (RP)‐encoding genes is heavily dependent upon the TFIID complex, composed of the TATA Binding Protein and 14 associated Tafps. RP gene transcription is also directly influenced by the DNA binding transcription factor Repressor Activator Protein (Rap1p). Recently we showed that Rap1p and TFIID engage in direct interaction, this is important for transcription of RP genes. We have used Far Western Protein Blotting coupled with truncation mutagenesis to identify the regions responsible for Rap1p‐TFIID interaction in all three Tafp subunits involved. Rap1p interacts with Taf4p through the conserved region between the canonical Histone Fold Domain (HFD) and the Concerved C‐Terminal Domain (CCTD). Rap1p contacts Taf5p through residues located in the highly conserved NTD regions. We confirmed regions involved are essential for cellular viability in both Taf4p and Taf5p. Consistent with this observation, using random mutagenesis we were able to generate large families of conditional mutants of both TAF4 and TAF5 linked to regions encoding specific domains of the proteins. We are now using these tools in systematic molecular genetic and biochemical dissection of this interaction. This study will allow us to characterize Rap1p‐TFIID interaction in much greater detail.