The C-terminal domain (CTD) of RNA polymerase II (Pol II) consists of conserved heptapeptide repeats, which undergo a pattern of phosphorylations during the transcription cycle. Although phosphorylation of Tyr1 of human Pol II was first described almost two decades ago, its functional significance has remained unknown. By developing a phosphorylated Tyr1-specific antibody, Mayer et al. were able to demonstrate that transcribing budding yeast Pol II CTD is phosphorylated at Tyr1, in addition to Ser2, Thr4, Ser5 and Ser7, and is correlated with mRNA expression. Tyr1 phosphorylation increases downstream of the transcription start sites of transcribed genes and decreases before the polyadenylation (pA) site. Genomic chromatin-immunoprecipitation profiles of CTD-binding termination factors Rtt103 and Pcf11 peaked downstream of the pA site, whereas that of Nrd1 is consistent with its binding to Ser5-phosphorylated CTD, which suggests that Tyr1 phosphorylation may impair CTD binding of these termination factors. Indeed, none of the termination factors bound to Tyr1-phosphorylated CTD peptide in vitro, consistent with this idea. Structural modeling further revealed that Tyr1 phosphorylation causes steric clashes that block the CTD interaction and showed that Thr4 phosphorylation also destabilizes the interaction with CTD-binding termination factors, which is consistent with low Thr4 phosphorylation levels at the pA sites of genes. Finally, the CTD-interacting domain of elongation factor Spt6 binds Ser2-phosphorylated CTD, and given the similar ChIP profiles for Ser2 and Tyr1 phosphorylation, the latter is expected not to interfere with Spt6 binding. Indeed, in vitro binding studies showed that Tyr1-phosphorylated CTD peptide stimulates Spt6 binding. So Tyr1-phosphorylated CTD triggers and blocks factor recruitment, thereby expanding the Pol II CTD code. (Science 336, 1723–1725, 2012)