DOA4 IN RELEASING UBIQUITIN FROM UBIQUITIN PROTEIN CONJUGATES

酿酒酵母的spt4和spt5基因分别为 在几次突变体搜索中发现了参与 转录调控。最新的生化和遗传分析 Spt4和Spt5已经表明，这些蛋白质在调节 RNA聚合酶II的加工性，很可能是通过管理 染色质过度转录基因的结构。生化 实验表明，Spt4和Spt5形成紧密的复合体。使用 标记了FLAG表位的Spt5的衍生物，我们有部分 纯化了Spt4/Spt5复合体。与这一观点一致的是 Spt4/Spt5复合体间歇性地与其他蛋白质相互作用 转录延伸的重复循环，还有其他几种 亚化学计量比Spt4/Spt5复合体与蛋白质共纯化 数量。在这些共同纯化的蛋白质中，Rpb1是最大的 RNA聚合酶亚基II。我们将亲和纯化Spt4/Spt5 并通过质谱学鉴定其他复制蛋白。 对这些蛋白质的鉴定应该能进一步深入了解 所需的机械的性质，以确保进程 RNA聚合酶II对转录本的延伸作用。

The SPT4 and SPT5 genes of Saccharomyces cerevisiae were identified in several mutant hunts for proteins involved in transcription regulation. Recent biochemical and genetic analysis of Spt4 and Spt5 has shown that these proteins play a role in regulating the processivity of RNA polymerase II, most likely by managing the structure of chromatin over transcribed genes. Biochemical experiments show that Spt4 and Spt5 form a tight complex. Using a derivative of Spt5 tagged with the Flag epitope, we have partially purified this Spt4/Spt5 complex. Consistent with the idea that the Spt4/Spt5 complex intermittently interacts with other proteins during the repetitive cycle of transcription elongation, several other proteins co-purify with the Spt4/Spt5 complex in substoichiometric quantities. Among these co-purifying proteins is Rpb1, the largest subunit of RNA polymerase II. We will affinity purify the Spt4/Spt5 complex, and identify other copufiying proteins by mass spectrometry. Identification of these proteins should provide further insight into the nature of the machinery that is required to insure processive elongation of transcripts by RNA polymerase II.