Chaperone-mediated ordered assembly of the SAGA and NuA4 transcription co-activator complexes

Abstract

Transcription initiation involves the coordinated activities of large multimeric complexes.Little is known about the mechanisms and pathways that drive their assembly from individual components. We report here several principles underlying the assembly of the highly conserved SAGA co-activator complex, which is composed of 19 subunits organized into functional modules. First, we demonstrate that SAGA assembles through an ordered pathway, in which the core subunit Spt20 recruits Tra1, which then promotes the incorporation of the de-ubiquitination module (DUB). Second, affinity purifications and phenotypic analyses identified a small region of Spt20 that is both necessary and sufficient to anchor Tra1 to SAGA. Third, Tra1 is shared with the NuA4 co-activator complex and is the only pseudokinase of the PIKK family. We accumulated functional and biochemical evidence that Tra1 shares a specific Hsp90 cochaperone with PIKKs, the Triple-T complex, for its cotranslational folding and de novo incorporation into both SAGA and NuA4. Finally, in contrast to its specific role in SAGA assembly, Tra1 contributes to scaffold the entire NuA4 complex. Overall, our study brings mechanistic insights into the de novo assembly of transcriptional complexes via ordered pathways and reveals the contribution of dedicated chaperones to this process.

New insights into the evolutionary conservation of the sole PIKK pseudokinase Tra1/TRRAP

“…Recently, we observed that the conditional loss of Tra2 disrupts NuA4 integrity and affects cell viability, indicating that Tra2 does indeed scaffold the assembly of the entire NuA4 complex [55]. In marked contrast, we showed that Tra1 has no scaffolding function within SAGA, but, rather, regulates the expression of a small subset of SAGA-dependent genes and specifically controls the incorporation of the DUB module [55].…”

“…Indeed, Tra1 is localized at the periphery of SAGA, which contacts the FAT domain through a surprisingly small and flexible region called the 'Hinge'. We recently demonstrated that Spt20 is the major interacting partner of Tra1 within SAGA, in both S. pombe and S. cerevisiae [55]. In contrast,…”

“…Phylogenetic analyses indicate that these paralogs result from a duplication of a single gene in the ancestor of the Schizosaccharomyces lineage, suggesting that each paralog diverged such that Tra1 and Tra2 are specific for each complex in S. pombe [19]. Recently, we observed that the conditional loss of Tra2 disrupts NuA4 integrity and affects cell viability, indicating that Tra2 does indeed scaffold the assembly of the entire NuA4 complex [55]. In marked contrast, we showed that Tra1 has no scaffolding function within SAGA, but, rather, regulates the expression of a small subset of SAGA-dependent genes and specifically controls the incorporation of the DUB module [55].…”

“…Importantly, TTT interacts genetically and physically with Tra1 in yeast [18,19,[73][74][75] and stabilizes TRRAP in human cells [61,[66][67][68]. Accordingly, we recently established that, in fission yeast, Hsp90 and TTT promote the de novo incorporation of Tra1 into SAGA and of Tra2 into NuA4 [55].…”

Structure of the transcription coactivator SAGA

“…Conserved residues are highlighted in blue. Key residues are labeled with asterisks 45 . Sc, Pp and Sp stand for Saccharomyces cerevisiae , Pichia Pastoris , and Schizosaccharomyces pombe , respectively.…”