Abstract
Repressor activator protein 1 (RAP1) assists GCN4-mediated HIS4 activation by overcoming some repressive aspect of chromatin structure to facilitate GCN4 binding. RAP1 also participates in other nuclear processes, and discrete domains of RAP1 have been shown to have specific properties including DNA binding, DNA bending, transcriptional activation, and silencing and telomere functions. To investigate whether specific domains of RAP1 are required to "open" chromatin and help GCN4 to activate the HIS4 gene, we examined the abilities of different truncated RAP1 proteins to perturb positioned nucleosomes via a nucleosomal RAP1 site in a yeast episome in vivo, and we tested HIS4 activation in yeast strains harboring truncated RAP1 mutants. We found that neither the DNA bending domain nor the putative activation domain of RAP1 is required for its ability to perturb the chromatin structure of a plasmid containing a RAP1 site. Similarly, neither the putative activation domain nor the N-terminal DNA-bending domain was required for GCN4-mediated activation of HIS4. We also used a rap1(ts) mutant to show that continuous occupancy of the HIS4 promoter by RAP1 is required for GCN4-mediated gene activation.
Highlights
Repressor activator protein 1 (RAP1)1 is an essential protein in yeast
When HIS4 activation is made to depend on other activators with a better ability to outcompete histones for binding to DNA, the RAP1 site becomes dispensable, indicating that the RAP1 requirement is a consequence of the poor ability of GCN4 to bind to chromatin under physiological conditions [10], More recently, we have used chromatin immunoprecipitation to show directly that GCN4 binding to the HIS4 promoter is reduced when the RAP1 binding site is mutated
Dispensability of the C-terminal and N-terminal Domains of RAP1 for Chromatin Perturbation and HIS4 Activation—We have examined the ability of truncated versions of RAP1, lacking different of its known functional domains, to perturb chromatin structure via a nucleosomal RAP1 binding site, and we find that neither the C-terminal part containing the silencing domain and the putative activation domain nor the N-terminal portion that is required for DNA bending by RAP1 is needed for RAP1-mediated chromatin perturbation
Summary
Repressor activator protein 1 (RAP1) is an essential protein in yeast. Binding sites for RAP1 have been found in promoters, silencers, and telomeres, and correspondingly, RAP1 participates in gene transcription, silencing, and telomere maintenance [1]. A property of RAP1 that is likely to contribute to its function in disparate processes is a potent ability to perturb chromatin structure in a way that increases access by DNA-binding proteins and nucleases, a property we refer to as “chromatin opening” [9, 10]. This property most clearly contributes to RAP1 function at the HIS4 promoter where a RAP1 binding site is required to overcome the repressive effect of chromatin structure and allow GCN4 to bind and activate transcription in response to conditions of amino acid starvation [10, 11].
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