Function of a Eukaryotic Transcription Activator during the Transcription Cycle

Abstract

Site-specific photocrosslinkers positioned within the central transcription-activating region of yeast Gcn4 were used to identify, in an unbiased way, three polypeptides in direct physical proximity to the activator during the process of transcription activation. Crosslinking was specific and did not change during different steps of the transcription cycle. The crosslinking targets were identified as Tra1, Gal11, and Taf12, subunits of four complexes (SAGA, NuA4, Mediator, and TFIID) known to play a role in gene regulation. Using this crosslinking assay, an activating region mutant, and extracts depleted of individual complexes containing the crosslinking targets, we found that contact with Tra1/SAGA is critical for activation, Gal11 contact has a modest effect on activation, and contact with TFIID and NuA4 is of little or no importance for activation under our conditions. Thus, a single activating region contacts multiple factors, and each contact makes differential contributions to transcriptional activation.