The 3D Structural Analysis Of Yeast SAGA Complex By Cryo-EM

Eukaryotic gene transcription by RNA polymerase II?Pol II?is highly regulated and fine-tuned by diverse epigenetic complexes,which often function as co-activators or co-repressors to modulate transcription in concert with sequence-specific transcription factors?TFs?.In Saccharomyces cerevisiae,the 1.8 MDa coactivator SAGA?Spt-Ada-Gcn5-Acetyltransferase?complex was reported to be required for genome-wide transcription by RNA Pol II,and proposed to function as a general cofactor.Yeast SAGA is composed of 19 subunits,which are categorized into four modules,namely HAT?Gcn5,Ada2,Ada3,Sgf29?,DUB?Ubp8,Sgf11,Sgf73,Sus1?,TAF?Taf5,Taf6,Taf9,Taf10,Taf12?and SPT?Tra1,Ada1,Spt3,Spt7,Spt8,Spt20?.Two catalytic subunits,Gcn5 within HAT1 and Ubp8 within DUB3,permit histone H3acetylation and ub-H2B deubiquitylation activities of the SAGA complex.Remarkably,acetylation and deubiquitylation carried out by SAGA are crucial for transcriptional initiation and elongation;in yeast,inactivation of SAGA leads to global decrease of RNA Pol II-mediated transcription.We successfully purified endogenous SAGA complex that contains all 19 subunits from fermented S.cerevisiae cells after optimization of growth conditions.The structure of SAGA was then determined by single particle analysis at a resolution up to 6.9?,with the largest component Tra1 solved at 4.6?.By contrast,lobe B is less well resolved,and an overall resolution of 9.3?was calculated at FSC=0.5.The two enzymatic modules,HAT and DUB,are located within Lobe B.The blurry density of lobe B likely reflects conformational dynamics of the catalytic modules in the absence of the nucleosomal substrate.In addition,compared to the previous published structure,we identified two new structural features in our map.Combining the CX-MS results,we proposed an improved model for yeast SAGA organizations:the Spt8 in R-right region and the Taf6 in R-left region.Focused on Tra1,the exposed positively charged surfaces are distinct between the Tra1_SAGA and Tra1_{Nu A4},which is also responsible for DNA binding and TFs recruitments.This evidence indicated that SAGA is unlikely to function corporately with Nu A4,even both are acetyltransferases.Finally,we docked ub-Nucleosome-DUB structure into the SAGA and found the ubiquitylated nucleosome was captured by the DUB module and swung away from the HAT module.It appears that the HAT and DUB modules could not engage in the catalysis of one nucleosome substrate concurrently.Our structural analysis suggests that the DUB and HAT modules of SAGA likely work sequentially,in which histone H3 acetylation by HAT is preceded by the ubiquitin removal and ub-nucleosome recognition by DUB.