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The primary transcriptional regulator of amino-acid biosynthesis in yeast is Gcn4. Increased amounts of this protein results in the de-repression of amino-acid biosynthetic pathways and the concomitant increase of amino-acid pools. Gcn4 is a typical modular transcriptional activator composed of a DNA binding domain of the b-Zip type, and an Òacidic activationÓ domain. Although the structure and function of the DNA binding domain is understood in considerable detail, both in vivo and in vitro, this is not the case for the activation domain. These domains are believed to interact either directly or through protein bridges (or both) with components of the basic transcriptional machinery, and consequently to increase the rate of formation of productive transcriptional initiation complexes. Biochemical evidence supports the direct interaction of acidic activation domains with TBP and TFIIB but their functional significance is questionable. In contrast, it has recently been shown that the support of transcriptional activation in vitro requires the mediation of a multisubunit complex. The requirement for such mediation is supported in vivo by genetic analysis. The function of the GCN4 activation domain, as well as of other transcription activation domains (GAL4, VP16), depends on the function of Gcn5, Ada2 and Ada3. These three proteins define one pathway of transcriptional activation, and evidence exists for their interaction. This pathway accounts for only part of the GCN4 activation domain function. We have defined a second pathway and have isolated a dominant mutation, AFR1, which defines a gene product involved in this pathway. AFR1,gcn5 double mutants are non-viable. Finally, by using asymmetric GCN4 DNA targets, we have established that Gcn4 binding on these targets uncovers a polarity for the requirement of the Gcn5-Ada2 pathway that depends on the relative to the TATA location of the optimal GCN4 half site. We interpret this to reflect a local spatial or conformational alteration of the activation domain.
Current objectives
Given this brief introduction, the main focus of ongoing research is to define precisely the structural and functional determinants of the Gcn4 activation domain as it relates to interactions with adaptors or stabilizers and components of the basic transcriptional machinery. Until now, such an analysis has suffered because of the lack of specific molecules that function as co-activators; thus any attempts to analyze activation domains was performed in this vacuum. The basic tools for this analysis are the Gcn5-Ada2 pathway, the AFR1 pathway, the target dependent polarity and general transcription factors. Understanding of molecular interactions will involve also studies to define these pathways more precisely in respect to their organization and specific contacts with the activation domains-basic transcription factors and other components involved in the generation of productive transcription initiation complexes
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