The role of the central region of Ada2 in gene regulation. Saccharomyces cerevisiae

The SAGA complex of Saccharomyces cerevisiae contains greater than 20 components that acetylate and deubiquitylate nucleosomal histones. Its acetyltransferase, Gcn5 preferentially acetylates histones H3 and H2B and is regulated through interactions with Ada2 and Ngg1/Ada3. The N-terminal region of Ada2 contains a SANT domain that contacts Gcn5 near its catalytic site. Sequence alignments of Ada2 homologues indicate a conserved ~120 amino acid residue central region that interacts with Ngg1.To examine the function of this central region, we constructed ada2 alleles with mutations of clustered conserved residues. One of these alleles, ada2-RLR, resulted in a ~3-fold reduction in transcriptional activation of the PHO5 gene and growth changes that parallel deletion of ada2. Microarray analyses further revealed that ada2-RLR alters expression of a subset of those genes affected by deletion of ada2. Keywords: yeast, Ada2, SAGA complex, gene expression, genetic modification Overall design: Three dye-swapped, biological replicate experiments were performed for BY4842 (ada2delta) with reference to BY4741 (wt). Similarly, in the BY4842 (ada2delta) strain background three dye-swapped, biological replicate experiments were performed for a strain expressing a mutant ada2 allele from a plasmid with reference to a strain expressing wild-type ada2 from a plasmid.

酿酒酵母（Saccharomyces cerevisiae）的SAGA复合物（SAGA complex）包含20余种组分，可催化核小体组蛋白的乙酰化与去泛素化修饰。其所含的乙酰转移酶Gcn5可优先乙酰化组蛋白H3与H2B，并通过与Ada2和Ngg1/Ada3的相互作用实现调控。Ada2的N端区域含有一个SANT结构域，能够在Gcn5的催化位点附近与其结合。对Ada2同源蛋白的序列比对结果显示，其存在一段长度约120个氨基酸残基的保守中央区域，该区域可与Ngg1发生相互作用。为探究该保守中央区域的功能，我们构建了携带簇状保守残基突变的ada2等位基因。其中一株突变体ada2-RLR可使PHO5基因的转录激活水平降低约3倍，其引发的生长表型变化与ada2基因缺失的结果一致。基因芯片（microarray）分析进一步表明，ada2-RLR可改变受ada2缺失影响的部分基因的表达水平。关键词：酵母，Ada2，SAGA复合物，基因表达，遗传修饰。整体实验设计：以BY4741（野生型，wt）为参照，对BY4842（ada2Δ）开展了3次生物学重复的染料互换实验。同样，在BY4842（ada2Δ）的菌株背景下，以携带质粒表达野生型ada2的菌株为参照，对携带质粒表达突变型ada2等位基因的菌株开展了3次生物学重复的染料互换实验。