‐Altered DNA‐Binding Specificity Transcription Activator Variant Provides Key To Unlocking Gene Activation Mechanism

Abstract

Transcription activator proteins confer target selective, gene‐specific activation in response to appropriate stimuli. In order to dissect the molecular mechanisms of gene transcription activation, it is imperative to understand the role(s) of the transcription activator. Unfortunately, a subset of DNA‐binding transcriptional activator proteins are resistant to study by the conventional means of fusing putative Activation Domains (ADs) to heterologous DNA Binding Domains (DBDs) to identify key transcription factor functional elements. The budding yeast Saccharomyces cerevisiae Repressor Activator Protein 1 (Rap1) is one such activator protein. Rap1 specifically binds over 500 DNA sites in the yeast genome, serving key roles in multiple cellular processes including: (a) telomere stabilization; (b) transcription repression of silent mating type loci; and (c) activation of two of the most vigorously transcribed classes of yeast genes: the SAGA‐dominated glycolytic enzyme‐encoding genes (GEs) and the TFIID‐dominated ribosomal protein genes (RPGs). However, fusions of Rap1 or Rap1‐domains with heterologous DBDs reportedly fail to robustly drive reporter gene expression.To enable study of the role of Rap1 in activation, an altered DNA‐binding specificity Rap1 variant (Rap1 AS) was generated using a direct screening/selection strategy in yeast wherein activation of a selectable HIS3 reporter gene depended upon the ability of DBD‐mutagenized variant forms of Rap1 to efficiently drive expression from a mutant Rap1 enhancer (UASRAP1). Multiple candidate putative Rap1 AS variants scored in this screen. One has been characterized in detail. In both biochemical (DNA gel mobility shift) and functional (HIS3 reporter gene expression) assays, Rap1 AS preferentially bound the selecting mutant UASRAP1, displaying a significant discrimination for mutant vs. wild‐type (WT) UASRAP1 sequences. Yeast pseudodiploid for Rap1 (Rap1 WT and Rap1 AS) grow like WT, showing that Rap1 AS does not interfere with the many essential functions of Rap1 WT. Subsequent mutagenesis experiments with Rap1 AS identified a putative activation domain that mapped to a ~40 amino acid region within the C‐terminal 1/3 of Rap1. Systematic point mutagenesis of this mapped AD has identified eight amino acids that contribute critically to AD function.The collection of Rap1 AD mutants will be used to investigate the transcription activation mechanisms used to drive the TFIID‐dominated RPGs. Since we have previously shown that Rap1 interacts directly with TFIID, and is the only RPG activator absolutely required for RPG transcription, mechanistic analyses performed with the Rap1 AD mutants should unlock the key step(s) to RPG activation. Because few TFIID‐dependent activation mechanisms have been carefully dissected, these key steps will likely reveal general principles of TFIID‐dependent gene activation applicable to all organisms.Support or Funding InformationThis research was supported by the NIH Molecular Endocrinology Training Program (METP) training grant (T32 DK07563) and the NIH R01 grants GM52461 and GM115892.