Recognition of the RNA polymerase II CTD by transcription termination factors

The phosphorylation state of the C-terminal domain (CTD) of RNA polymerase II controls the assembly of RNA processing and transcription factors on the elongating enzyme. I explored the molecular basis for the recognition of specific CTD phosphoisoforms by CTD-interacting domains by probing the transcription termination and 3'-end processing factors Rtt103 and Pcf11 with an exhaustive panel of CTD mimics containing all possible phosphorylation states. When presented with a single binding site composed of two heptad repeats, Rtt103 binds to Ser2 phosphorylated (Ser2P) peptide strongly and specifically, while Pcf11 does so much less well. A single amino acid change reverses the specificity of these two proteins in vitro and in vivo. However, both Pcf11 and Rtt103 cooperatively recognize peptides containing two or more binding sites. My data suggests that the cooperativity observed for Pcf11 is critical for its proper recruitment to bona-fide transcription termination sites, while preventing transcription at cryptic sites. My work demonstrates for the first time that neighboring CTD repeats can function together to create a precisely tuned switch that can achieve a rapid, sigmoidal response allowing for precise control of co-transcriptional RNA processing.;The phosphorylation state of the CTD is dynamically altered during transcription by a number of CTD kinases and phosphatases. I have performed a number of initial experiments on the CTD Ser5P phosphatase, Ssu72, in order to obtain a suitable amount of protein for structure determination. Using a combination of sequence alignment and structure prediction, I was able to produce a soluble yeast Ssu72 construct. Also, I was able to produce yeast Ssu72 co-expressed with its binding partner Pta1. In addition to my work on yeast Ssu72, I also have completed partial assignments of the human Ssu72 protein.