COMPONENTS OF HAT COMPLEXES IN TRANSCRIPTIONAL CONTROL USING HIV PROMOTER

核心组蛋白的N-末端赖氨酸的组蛋白乙酰化具有以下作用： 与转录调控有关。 我们的实验室最近 鉴定了四种不同的天然组蛋白乙酰转移酶（HAT） 酵母的复合物。 其中两种酶，1.8MDa佐贺复合物 和0.8kDa的Ada复合物都含有接头蛋白Ada 2， Ada 3和Gcn 5。 最初鉴定的共激活蛋白Gcn 5是 在佐贺内具有HAT活性主要亚基（如果不是唯一亚基的话 还有艾达为了描述和进一步调查 复合物，我们纯化这种HAT酶超过7个连续的不同 色谱柱。 然后分析高度纯化的级分 通过质谱法将肽与酵母基因组进行比较 序列数据库 有趣的是，几个身份不明的开放式阅读 框架被发现是Ada复合体的一部分，这表明了一个新的作用， 对于这些蛋白质。 我们目前正在生产酵母敲除 这些ORF的菌株，以进一步研究它们在 Ada HAT复合体 直接调查Ada和佐贺是否起作用 在转录调控中， 用HIV-1启动子进行转录测定。 地掌握这一 启动子已经在体内连接到乙酰化。 Ada和佐贺 复合物刺激HIV-1从预组装的核小体转录 以乙酰辅酶A依赖的方式。 此外，对于Ada，我们 发现只有与HIV-1相关的组蛋白的乙酰化 模板可以促进转录。

Histone acetylation of N-terminal lysines of the core histones has been linked to transcriptional regulation. Our lab has recently identified four distinct native histone acetyltransferase (HAT) complexes from yeast. Two of these enzymes, the 1.8MDa SAGA complex and the 0.8kDa Ada complex both contain the adaptor proteins Ada2, Ada3 and Gcn5. The originally identified coactivator protein Gcn5 is the primary if not only subunit possessing HAT activity within SAGA and Ada. In an attempt to characterize and further investigate the Ada complex, we purified this HAT enzyme over 7 consecutive different chromatographic columns. Highly purified fractions were then analysed by mass spectrometry and peptides were compared to the yeast genome sequence database. Interestingly, several unidentified open reading frames were found to be part of the Ada complex suggesting a new role for these proteins. We are currently generating yeast knock-out strains of these ORFs to further investigate their function within the Ada HAT-complex. Direct investigation whether Ada and SAGA function in transcriptional regulation was done by performing in vitro transcription assays with the HIV-1 promoter. Regulation of this promoter has been linked in vivo to acetylation. The Ada and SAGA complexes stimulate HIV-1 transcription from pre-assembled nucleosomal templates in an acetylCoA-dependent manner. Furthermore, for Ada we find that acetylation of only the histones associated with the HIV-1 template can facilitate transcription.