The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae. First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24–26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1. In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length.

The C‐terminal domain (CTD) of RPB1, the largest subunit of RNA polymerase II is comprised of repeating units of a seven amino acid sequence and is indispensible for function in eukaryotes. The aim of this study was to determine if repeats had specific function, dependent on their location within the sequence, or whether repeats were largely redundant. Using a library of precisely designed CTD mutants, a novel TET‐off system to control RPB1 expression, and phenotypic analysis in yeast we demonstrate that, in fact, different regions of the CTD exhibit different function. Specifically, we show differential sensitivity to various transcriptional inhibitors and can link phenotypes to the presence or absence of phosphorylatable serine residues. This study leads to a novel model for the assembly of CTD‐associating factors on the CTD during transcription. We also begin to use this library to identify changes in CTD‐associating protein recruitment to the CTD and to identify regions of the CTD important for processes such as mRNA processing and histone methylation.

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