In different phases of the transcription cycle, RNA polymerase (Pol) II recruits various factors via its C-terminal domain (CTD), which consists of conserved heptapeptide repeats with the sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. We show that the CTD of transcribing yeast Pol II is phosphorylated at Tyr1, in addition to Ser2, Thr4, Ser5, and Ser7. Tyr1 phosphorylation stimulates binding of elongation factor Spt6 and impairs recruitment of termination factors Nrd1, Pcf11, and Rtt103. Tyr1 phosphorylation levels rise downstream of the transcription start site and decrease before the polyadenylation site, largely excluding termination factors from gene bodies. These results show that CTD modifications trigger and block factor recruitment and lead to an extended CTD code that explains transcription cycle coordination on the basis of differential phosphorylation of Tyr1, Ser2, and Ser5.

The eukaryotic RNA polymerases Pol I, II, and III use different promoters to transcribe different classes of genes. Promoter usage relies on initiation factors, including TFIIF and TFIIE, in the case of Pol II. Here, we show that the Pol I-specific subunits A49 and A34.5 form a subcomplex that binds DNA and is related to TFIIF and TFIIE. The N-terminal regions of A49 and A34.5 form a dimerization module that stimulates polymerase-intrinsic RNA cleavage and has a fold that resembles the TFIIF core. The C-terminal region of A49 forms a "tandem winged helix" (tWH) domain that binds DNA with a preference for the upstream promoter nontemplate strand and is predicted in TFIIE. Similar domains are predicted in Pol III-specific subunits. Thus, Pol I/III subunits that have no counterparts in Pol II are evolutionarily related to Pol II initiation factors and may have evolved to mediate promoter specificity and transcription processivity.

The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y1-S2-P3-T4-S5-P6-S7. Dynamic phosphorylation of the CTD coordinates Pol II progression through the transcription cycle. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y1, S2, T4, S5, and S7 in mammalian and yeast cells. Although specific phosphorylation signatures dominate, adjacent CTD repeats can be differently phosphorylated, leading to a high variation of coexisting phosphosites in mono- and di-heptad CTD repeats. Inhibition of CDK9 kinase specifically reduces S2 phosphorylation levels within the CTD.