Abstract
Repressor activator protein 1 (Rap1) performs multiple vital cellular functions in the budding yeast Saccharomyces cerevisiae These include regulation of telomere length, transcriptional repression of both telomere-proximal genes and the silent mating type loci, and transcriptional activation of hundreds of mRNA-encoding genes, including the highly transcribed ribosomal protein- and glycolytic enzyme-encoding genes. Studies of the contributions of Rap1 to telomere length regulation and transcriptional repression have yielded significant mechanistic insights. However, the mechanism of Rap1 transcriptional activation remains poorly understood because Rap1 is encoded by a single copy essential gene and is involved in many disparate and essential cellular functions, preventing easy interpretation of attempts to directly dissect Rap1 structure-function relationships. Moreover, conflicting reports on the ability of Rap1-heterologous DNA-binding domain fusion proteins to serve as chimeric transcriptional activators challenge use of this approach to study Rap1. Described here is the development of an altered DNA-binding specificity variant of Rap1 (Rap1AS). We used Rap1AS to map and characterize a 41-amino acid activation domain (AD) within the Rap1 C terminus. We found that this AD is required for transcription of both chimeric reporter genes and authentic chromosomal Rap1 enhancer-containing target genes. Finally, as predicted for a bona fide AD, mutation of this newly identified AD reduced the efficiency of Rap1 binding to a known transcriptional coactivator TFIID-binding target, Taf5. In summary, we show here that Rap1 contains an AD required for Rap1-dependent gene transcription. The Rap1AS variant will likely also be useful for studies of the functions of Rap1 in other biological pathways.
Highlights
Transcription activation is the first step in the carefully orchestrated process of eukaryotic protein-coding gene expression [1]
The RAP1 sequences encoding the Repressor activator protein 1 (Rap1) amino acid residues predicted by X-ray crystallography to contact the key mutationally sensitive UASRap1 DNA bps were subjected to site-directed mutagenesis
Plasmids carrying these mutant RAP1 variants were introduced into yeast carrying an integrated reporter gene whose expression was driven by the inactivated mutant UASRap1 enhancer
Summary
Transcription activation is the first step in the carefully orchestrated process of eukaryotic protein-coding gene expression [1]. Together the transcription machinery and coregulators clearly play essential roles in mRNA biosynthesis, it is the enhancer DNA-binding transcriptional activator proteins that confer gene specificity to transcriptional control by selectively responding to discrete cell-external and/or cell-internal molecular cues [9]. Upon receiving the signal for gene activation, a transcriptional activator protein binds to its specific and accessible enhancer target recognition site in chromatin through its DBD. These enhancer DNA-bound proteins transmit a signal for activation, via their AD, which either directly or indirectly results in the activation of the mRNA gene transcription machinery on the promoter of the cis-linked target gene [10]. Studies that define the structure and function of transactivator ADs, and their coactivator targets, will provide critical tools for developing deeper insight into the molecular mechanisms that regulate specific target gene transcription
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