Abstract
Transcription initiation involves the coordinated activities of large multimeric complexes, but little is known about their biogenesis. Here we report several principles underlying the assembly and topological organization of the highly conserved SAGA and NuA4 co-activator complexes, which share the Tra1 subunit. We show that Tra1 contributes to the overall integrity of NuA4, whereas, within SAGA, it specifically controls the incorporation of the de-ubiquitination module (DUB), as part of an ordered assembly pathway. Biochemical and functional analyses reveal the mechanism by which Tra1 specifically interacts with either SAGA or NuA4. Finally, we demonstrate that Hsp90 and its cochaperone TTT promote Tra1 de novo incorporation into both complexes, indicating that Tra1, the sole pseudokinase of the PIKK family, shares a dedicated chaperone machinery with its cognate kinases. Overall, our work brings mechanistic insights into the assembly of transcriptional complexes and reveals the contribution of dedicated chaperones to this process.
Highlights
Transcription initiation involves the coordinated activities of large multimeric complexes, but little is known about their biogenesis
S. pombe offers a unique opportunity to study the specific contribution of the Triple-T complex (TTT) and Tra[1] to SAGA and NuA4 organisation and function
We analysed SAGA purifications from RI-tra[1] tel2-auxin-inducible degron (AID) cells using silver staining (Fig. 3b) and quantitative mass spectrometry (MS) analyses (Fig. 3d). Both approaches showed decreased interaction between newly synthesised Tra[1] and affinity purified Spt[7] in cells partially depleted of Tel[2]. These results demonstrate that TTT contributes to the de novo incorporation of Tra[1] into the SAGA complex
Summary
Transcription initiation involves the coordinated activities of large multimeric complexes, but little is known about their biogenesis. Chromatin-modifying and -remodelling complexes often share functional modules and probably require dedicated mechanisms and chaperones for their proper assembly[2]. One such complex, the Spt-Ada-Gcn[5] acetyltransferase (SAGA) co-activator, bridges promoter-bound activators to the general transcription machinery. Genetic and biochemical studies indicate that Tra[1] primary role is to mediate the transactivation signal from activators by recruiting SAGA and NuA4 to chromatin It has been difficult, to delineate the specific contribution of Tra[1] to SAGA and NuA4 architecture and activities because, to date, no clear separation-of-function alleles exist. Little is known about how Tra[1] incorporates into the SAGA and NuA4 complexes, whether it involves similar or distinct mechanisms, and which chaperone or assembly factors are required
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