Subject(s)
N-Myc Proto-Oncogene Protein/metabolism , DNA Breaks, Double-Stranded , DNA Damage , DNA Replication , Exosome Multienzyme Ribonuclease Complex/genetics , Exosome Multienzyme Ribonuclease Complex/metabolism , Humans , N-Myc Proto-Oncogene Protein/chemistry , N-Myc Proto-Oncogene Protein/genetics , Neurons/metabolism , RNA Polymerase II/metabolism , Transcription Termination, Genetic , Transcriptional Elongation Factors/metabolismSubject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA/metabolism , Translocation, Genetic/genetics , Binding Sites , Chondroitin Sulfate Proteoglycans/metabolism , DNA-Binding Proteins/metabolism , Humans , Nucleic Acid Conformation , Transcription, Genetic/genetics , CohesinsABSTRACT
Targets of the tandem Gcn4 acidic activation domains in transcription preinitiation complexes were identified by site-specific cross-linking. The individual Gcn4 activation domains cross-link to three common targets, Gal11/Med15, Taf12, and Tra1, which are subunits of four conserved coactivator complexes, Mediator, SAGA, TFIID, and NuA4. The Gcn4 N-terminal activation domain also cross-links to the Mediator subunit Sin4/Med16. The contribution of the two Gcn4 activation domains to transcription was gene specific and varied from synergistic to less than additive. Gcn4-dependent genes had a requirement for Gal11 ranging from 10-fold dependence to complete Gal11 independence, while the Gcn4-Taf12 interaction did not significantly contribute to the expression of any gene studied. Complementary methods identified three conserved Gal11 activator-binding domains that bind each Gcn4 activation domain with micromolar affinity. These Gal11 activator-binding domains contribute additively to transcription activation and Mediator recruitment at Gcn4- and Gal11-dependent genes. Although we found that the conserved Gal11 KIX domain contributes to Gal11 function, we found no evidence of specific Gcn4-KIX interaction and conclude that the Gal11 KIX domain does not function by specific interaction with Gcn4. Our combined results show gene-specific coactivator requirements, a surprising redundancy in activator-target interactions, and an activator-coactivator interaction mediated by multiple low-affinity protein-protein interactions.