Identification of a domain within human TAF(I)48, a subunit of Selectivity Factor 1, that interacts with helix 2 of TBP

• Published in: Gene Vol. 338; no. 2; pp. 177 - 186
• Main Authors: Xu, Shuping, Hori, Roderick T
• Format: Journal Article
• Language: English
• Published: Netherlands 01.09.2004
• Subjects: Amino Acid Sequence; Binding Sites - genetics; Humans; Immunoblotting; Models, Molecular; Molecular Sequence Data; Mutation; Pol1 Transcription Initiation Complex Proteins - chemistry; Pol1 Transcription Initiation Complex Proteins - genetics; Pol1 Transcription Initiation Complex Proteins - metabolism; Protein Binding; Protein Structure, Tertiary; Saccharomyces cerevisiae - genetics; TATA-Box Binding Protein - chemistry; TATA-Box Binding Protein - genetics; TATA-Box Binding Protein - metabolism; Two-Hybrid System Techniques
• ISSN: 0378-1119
• DOI: 10.1016/j.gene.2004.04.034

RNA polymerase I transcription in human cells requires Selectivity Factor 1, a multisubunit complex composed of the TATA-box-binding protein (TBP) and three TBP-associated factors (TAFs) called TAF(I)48, TAF(I)63 and TAF(I)110. Each of the Selectivity Factor 1 subunits binds directly to the other three components, but these interactions have not been characterized. This study is the initial identification and analysis of a TBP-binding domain within a Selectivity Factor 1 TAF. The interaction between human TBP and human TAF(I)48 was initially examined using the yeast two-hybrid assay, and a TBP-binding domain was identified in the carboxyl-terminus of human (h)TAF(I)48. Consistent with this result, the hTAF(I)48 carboxyl-terminus was able to bind directly to TBP in protein-protein interaction assays. When mutations were introduced into the hTAF(I)48 carboxyl-terminus, we identified changes in uncharged and positive residues that affect its interaction with TBP. By examining TBP mutants, residues within and adjacent to helix 2 of TBP, previously demonstrated to interact with subunits of other TBP-containing complexes [Transcription Factor IID (TFIID) and TFIIIB] were also found to diminish its affinity for the carboxyl-terminus of hTAF(I)48. The regions of hTAF(I)48 and TBP that interact are compared to those identified within other complexes containing TBP.