RNA polymerase II (RNAP II) is responsible for transcribing messenger RNAs (mRNAs) as well as non-coding RNAs such as small nuclear RNAs (snRNAs) and microRNAs in eukaryotic cells. Rpb1, the largest catalytic subunit of this complex, possesses a unique C-Terminal Domain (CTD) that consists of tandem heptad repeats (the number varying from 26 to 52 by organism) with the consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser (Y1S2P3T4S5P6S7). The CTD is extensively phosphorylated and dephosphorylated on non-proline residues during different steps of the transcription cycle, with roles for the threonine (Thr4) and tyrosine (Tyr1) attracting more attention. For example, in chicken cells, Thr4 functions in histone mRNA 3’ end formation, and Tyr1 phosphorylation is primarily associated with promoters and upstream antisense RNA formation, as well as preventing degradation of the polymerase, processes not found across all eukaryotes. A detailed introduction is described in Chapter 1.
Taking advantage of the genetic tractability of yeast cells, we created a yeast (S. cerevisiae) strain with all CTD threonines substituted with valines (T4V) to study the role of CTD Thr4 in transcription in yeast, which prior to this study has been poorly characterized in S. cerevisiae. Using the T4V strain, we found that Thr4 was required for proper transcription of phosphate-regulated (PHO) and galactose-inducible (GAL) genes. We found genetic links between the T4V polymerase and genes encoding subunits of the Swr1 and Ino80 chromatin remodeling complexes, as well as the histone variant Htz1. We further provide evidence that CTD Thr4 is required for proper eviction of Htz1 by the Ino80 complex from genes requiring Thr4 for activation, presented in Chapter 2 of this thesis.
Finally, Chapter 3 describes the functions of CTD Tyr1 in S. cerevisiae. Using a strategy similar to the T4V strain, I created a strain expressing an endogenous Rpb1 with all CTD tyrosine residues mutated to phenylalanine (Y1F). We found that this strain was viable, but with a severe slow-growth phenotype. We found genetic links between the Y1F polymerase and kinase/cyclin pair Srb10/Srb11, as well as an increase in occupancy on chromatin for the same. Further analysis indicated that RNA levels of genes associated with MAP Kinase associated stressors were dysregulated, and poly(A) site selection was biased towards distal poly(A) sites. Next, using an in vitro kinase assay, we showed Tyr1 phosphorylation on the CTD by MAP kinase Slt2, and in vivo CTD Tyr1 phosphorylation levels changed based on Slt2-associated stress response, as well as a decrease in in vivo Tyr1P-RNAP II from an Slt2 kinase-dead strain. Analysis of termination factors Nrd1 and Rtt103 showed transcription termination defects were likely the result of disruption of the interaction between the CTD interacting domains of these two proteins and the Y1F CTD. Extending this, we found additional disruptions in Slt2 recruitment to chromatin, increasing the depth of our knowledge of the interplay between induction of stress-associated genes, Slt2 function, and Nrd1-mediated termination.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/D8ST828W |
| Date | January 2017 |
| Creators | Yurko, Nathan Michael |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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