RNA Polymerase II CTD Tyrosine 1 is Required for Efficient Termination by the Nrd1-Nab3-Sen1 Pathway (ChIP-chip)

Transcription termination is key to gene regulation as it prevents transcription interference with neighboring genes. In Saccharomyces cerevisiae, termination at protein-coding genes is coupled to the cleavage of the nascent transcript, while most non-coding RNA transcription relies on a cleavage-independent termination pathway involving Nrd1, Nab3 and the helicase Sen1 (NNS pathway). In both pathways, the recruitment of termination factors involves phosphorylated forms of the RNA polymerase II C-terminal domain (CTD) but the contribution of individual CTD residues was never systematically investigated. Here, we investigated the impact of mutating phosphorylation sites in the CTD on termination. We observed widespread termination defects at protein-coding genes in mutants for Ser2 or Thr4 but rare defects in Tyr1 mutants for this class of genes. Instead, mutating Tyr1, or its phosphatase Glc7, led to widespread termination defects at non-coding genes known to terminate via the NNS pathway. These defects can be suppressed by slowing down transcription, suggesting that Tyr1 mediates termination via the regulation of elongation or pausing. Our work redefines the role of Tyr1 in termination at protein-coding genes in budding yeast and highlights its key role in termination by the NNS pathway. We took a systematic approach to examine the genome-wide distribution (using ChIP-chip) of various RNAPII CTD mutants. First, ChIPs were performed in a dual-tag system, where an ectopic rpb1 allele —carrying a desired CTD mutation—, tagged Flag, is expressed from a centromeric plasmid under the control of the RPB1 promoter in presence of the endogenous RPB1 allele tagged Myc. Both the mutant (Flag) and the endogenous WT (Myc) Rpb1 were assayed by ChIP-chip from the same extract, allowing direct comparison between the occupancy profile of RNAPII carrying a CTD mutation with that of a WT RNAPII. ChIPs (Cy5) were hybridyzed against Input (Cy3). Second, the project includes Pcf11, Rtt103, Nrd1 and Sen1 ChIP-chip in the various RNAPII CTD mutant cells where the endogenous Rpb1-FRB is anchored-away with the help of a rapamycin treatment. ChIPs (Cy5) were hybridized against a ChIP sample from an isogenic non-tagged strain (Cy3). Third, Rpb3 was profiled in WT, ctk1Δ, endo-S2A, sen1-R308W, nrd1ΔCID and rpb1-G1097D mutants and in cells where Nrd1 or Glc7 were anchored-away from the nucleus. ChIPs (Cy5) were hybridyzed against Input (Cy3). Finally, Tyr1-P was profiled in WT cells. ChIPs (Cy5) were hybridyzed against Input (Cy3). All ChIP-chip experiments were done in duplicates. Microarray data were normalized using the Lima Loess method and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).