Identification and characterization of novel protein-protein interactions with the basal transcription factor, TATA-binding protein

Recruitment of the TATA-Binding Protein, TBP, to the promoter is a critical, rate-limiting step that drives the initial phase of nearly all gene transcription events. Because of this, many transcriptional regulators target TBP, either to localize TBP at the promoter, or to relay signals between other promoter-bound protein complexes and the basal transcription machinery. Studies described herein were designed to identify novel protein-protein interactions with TBP. To do this, we screened mid-gestational pregnancy-associated cDNA prey libraries using two different yeast two-hybrid systems. Screens in both systems revealed both known and novel TBP interactors. B’-Related Factor 1 and Transcription Factor II A were identified in screens that used full-length TBP as bait. These proteins are known to interact with TBP and thus validated our system. In addition to known interactors, novel interactions with both the N-terminal (TBP-N) and C-terminal (TBP-C) domains of TBP were identified. CoactivatorAssociated Arginine Methyltransferase 1 (CARM1), Pax Transactivation DomainInteracting Protein (PTIP), and Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Factor (LITAF) all interacted with TBP-N. Proteins that interacted with TBP-C were Huntingtin-Associated Protein 1 (HAP1), four members of the Protein Inhibitor of Activated STAT (PIAS) family of transcriptional regulatory proteins, and Zinc Finger Protein 523 (ZFP523). The TBP interaction domains on PIAS1 and HAP1 were mapped to further define each interaction. Mapping studies revealed that TBP interacts with a single region on PIAS1, and with two separate regions on HAP1. We also show that TBP co-precipitates with PIAS1, PIAS3, HAP1, and PTIP. In conclusion, our studies, in agreement with previous published data suggest that TBP interacts with many classes of regulatory proteins, including transcriptional activators, repressors, and individual components of the transcriptional co-regulatory complexes. They also support the hypothesis that the TBP N-terminus is a protein interaction module and may provide clues to the function of the vertebrate-specific N terminus of TBP. 1 THE TATA-BINDING PROTEIN AND TRANSCRIPTION TATA-Binding Protein and Transcription Initiation Introduction to Transcription Transcription in eukaryotes is an ordered set of processes orchestrated mainly by large, multimeric protein complexes. The purpose of transcription is to copy the genetic information from the chromosomal DNA into a messenger (m) RNA. mRNA transcripts are templates used by the cellular translation machinery to make proteins. Throughout transcription initiation, elongation, and termination, the assembly and disassembly of subsets of polypeptides regulate a progression of events that occur only as the previous step is completed. Transcription relies on the combinatorial usage of both DNA (cisacting) and protein (trans-acting) factors to assure proper temporal and spatial expression of the genome. Since the focus of my studies is mammalian TATA-binding protein (TBP), a central player in transcription initiation, this review will revolve around the metazoan-specific transcription factors that are intimately involved in the processes underlying transcription initiation.