GROWTH-RELATED CHANGES IN YEAST CTD PHOSPHORYLATION

本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磷酸化在体内是必不可少的，但这种修饰起什么作用尚不清楚。我们以前已经表明，丝氨酸2和5的共识序列YSPTSPS在体外磷酸化。这些位点在体内的突变在酵母中是致命的。最近，我们已经使用特异性的单克隆抗体，这些磷酸化的残基，以显示酵母CTD磷酸化的模式在酵母生长周期的变化。本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一个以上的CTD磷酸异构体。其次，我们假设不同的CTD磷酸化异构体在转录中发挥不同的作用。为了验证这些假设，我们将：1。确定酵母CTD磷酸化在不同生长阶段的体内模式。我们将（1a）使用CTD结合试剂分离不同的pol II磷酸异构体。对于每种磷酸异构体，我们将确定：（1b）CTD上磷酸盐的数量，（1c）共有重复序列中哪些残基被磷酸化，以及（1d）磷酸盐在多个重复序列中的分布。2.表征单克隆抗体H5识别的pol II磷酸化异构体的产生和功能作用。具体地，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S2的磷酸化是否是诱导生长调节基因所必需的。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作用联系起来。这将是一个重要的贡献，在实现我们的长期目标，了解CTD的功能。了解这些途径以及它们如何调节酵母中的CTD磷酸化将进一步深入了解多细胞生物体中细胞的生长和非增殖状态之间的转变。

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.