Upstream activation sequence function in Saccharomyces cerevisiae: The potential for long distance transcriptional activation and the role of Sin4.

摘要

Upstream activation sequences (UASs) of Saccharomyces cerevisiae are promoter elements positioned 5' of the TATA box that serve as binding sites for transcriptional activators. Gene specific activators such as Gal4 and Gcn4 bind to UASs and interact with transcriptional co-activators and RNA polymerase II holoenzyme to mediate transcriptional activation. UASs are usually positioned 100-300 base pairs upstream of the TATA box and are generally unable to activate transcription if positioned more than 600 base pairs upstream of the TATA. This inability to activate "at a distance" is in striking contrast to enhancer elements of larger eukaryotes, which are often able to function several kilobases away from the basal promoter. To learn more about the transcriptional changes that occur when a UAS is positioned at a large distance from a TATA, we constructed a set of reporter strains with Gal4 binding sites located at a variable distance 5' of the HIS3 gene. These reporter strains were used in several screens and selections to identify factors that regulate long distance transcriptional activation in yeast. We isolated mutations in four loci that generally function to repress long distance activation: SIN4, SPT2, SPT10, and HTA1-HTB1. Double mutant analysis revealed that the SAGA and Mediator co-activator complexes are required for long distance transcriptional activation in sin4Δ mutants. Additionally, there is a modest increase in histone H3 acetylation over the reporter gene TATA box. Finally, truncation analysis of Sin4 identified a 50 amino acid region in the C-terminus of the protein important for its function and association with the Mediator complex. Taken together, our data have extended our understanding of both UAS function and the role of Sin4 in long distance activation.