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.
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