IDENTIFYING SUBSTRATES OF CYCLIN DEPENDENT KINASES BY SUBSTRATE TRAP & PURIFICAT

通过底物陷阱识别细胞周期依赖性激酶的底物

• 批准号: 6348283
• 负责人: JOHN R YATES
• 金额: $ 0.33万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6348283
• 关键词: AIDS; bacteria; bioengineering /biomedical engineering; biomedical resource; enzymes; immunology; infection; inflammation; lymphatic system; plants; proteins; virus

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.

核心组蛋白 N 末端赖氨酸的组蛋白乙酰化具有 与转录调控有关。 我们实验室最近 鉴定出四种不同的天然组蛋白乙酰转移酶 (HAT) 来自酵母的复合物。 其中两种酶，1.8MDa SAGA 复合物 和 0.8kDa Ada 复合物均含有接头蛋白 Ada2， Ada3 和 Gcn5。 最初鉴定的辅激活蛋白 Gcn5 是 SAGA 内具有 HAT 活性的主要（如果不是唯一）亚基 和艾达。试图描述并进一步研究 Ada 复杂的，我们纯化了这种 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 通过质谱分析将肽与酵母基因组进行比较 序列数据库。 有趣的是，几个不明身份的开放阅读 框架被发现是 Ada 复合体的一部分，暗示了一个新的角色 对于这些蛋白质。 我们目前正在产生酵母敲除 这些 ORF 的菌株，以进一步研究它们在 Ada HAT 复合体。 直接考察Ada和SAGA是否功能 转录调控是通过体外进行 HIV-1 启动子的转录测定。 对此的规定 启动子在体内与乙酰化有关。 艾达和SAGA 复合物刺激预组装核小体的 HIV-1 转录 以乙酰辅酶A依赖性方式模板。 此外，对于艾达，我们 发现仅与 HIV-1 相关的组蛋白发生乙酰化 模板可以促进转录。