GROWTH-RELATED CHANGES IN YEAST CTD PHOSPHORYLATION

我们研究的长期目标是了解RNA聚合酶II最大亚基的重复C末端结构域(CTD)的功能作用。CTD在体内是必不可少的，但这种修饰起什么作用尚不清楚。我们之前已经证明了YSPTSPS共有序列中的丝氨酸2和5在体外是磷酸化的。这些位点在体内的突变在酵母中是致命的。最近，我们使用了针对这些磷酸化残基的单抗来显示酵母CTD磷酸化模式在酵母生长周期中的变化。该提案中描述的实验集中在两个中心假设上。首先，我们假设酵母细胞含有不止一种CTD磷酸异构体。其次，我们假设不同的CTD磷酸异构体在转录过程中发挥不同的作用。为了验证这些假设，我们将：1.确定酵母CTD在不同生长阶段的体内磷酸化模式。我们将(1a)使用CTD结合试剂来分离不同的PolII磷酸异构体。对于每个磷酸异构体，我们将确定：(1B)CTD上的磷酸盐数量，(1C)共识重复内哪些残基被磷酸化，以及(1D)磷酸盐在多个重复中的分布。2.鉴定单抗H5识别的PolII磷酸异构体的产生和功能。具体地说，我们将(2a)确定CTDK-I产生H5反应性表位的作用。我们将(2b)确定CTDK-I在产生H5反应性表位中的作用。我们将(2b)确定是否需要S2的磷酸化来诱导生长调节基因。最后，我们将(2c)确定H5反应性磷酸异构体是否优先与生长调节基因相关。实现这两个目标将使我们能够将特定的CTD磷酸化状态与信号转导途径和基因调控中的特定角色联系起来。这将是对实现我们了解贸发会议职能的长期目标的重要贡献。了解这些途径以及它们如何调节酵母中CTD的磷酸化，将进一步深入了解多细胞生物体中细胞生长状态和非增殖状态之间的转换。

The long term objective of our research is to understand the functional role of the repetitive C-terminal domain (CTD) of the largest subunit of RNA polymerase II. CTD phosphorylation is essential in vivo but what role this modification plays is not known. We have previously shown that serines two and five in the consensus sequence YSPTSPS are phosphorylated in vitro. Mutation of these sites in vivo is lethal in yeast. More recently we have used monoclonal antibodies specific to these phosphorylated residues to show that the pattern of yeast CTD phosphorylation changes during the yeast growth cycle. The experiments described in this proposal focus on two central hypotheses. First, we hypothesize that yeast cells contain more than one CTD phosphoisomer. Second, we hypothesize that the different CTD phosphoisomers play different roles in transcription. To test these hypotheses we will: 1. Determine the in vivo pattern of yeast CTD phosphorylation at different stages of growth. We will (1a) use CTD-binding reagents to separate different pol II phosphoisomers. For each phosphoisomer we will determine: (1b) the number of phosphates on the CTD, (1c) which residues within the consensus repeat are phosphorylated, and (1d) the distribution of phosphates among the multiple repeats. 2. Characterize the generation and functional role of the pol II phosphoisomer recognized by monoclonal antibody H5. Specifically we will (2a) determine the role of CTDK-I is generating the H5-reactive epitope. We will (2b) determine the role of CTDK-I in generating the H5- reactive epitope. We will (2b) determine whether phosphorylation of S2 is required for induction of growth-regulated genes. Finally, we will (2c) determine whether H5-reactive phosphoisomer is preferentially associated with growth-regulated genes. Achieving these two aims will enable us to relate a specific CTD phosphorylation state to a signal transduction pathway and a specific role in gene regulation. This will be an important contribution in reaching our long term goal of understanding the function of the CTD. Understanding such pathways and how they may modulate CTD phosphorylation in yeast will field further insight into the transition between growth and non- proliferative states of cells in multicellular organisms.