We present genome-wide occupancy profiles for RNA polymerase (Pol) II, its phosphorylated forms and transcription factors in proliferating yeast. Pol II exchanges initiation factors for elongation factors during a 5′ transition that is completed 150 nucleotides downstream of the transcription start site (TSS). The resulting elongation complex is composed of all the elongation factors and shows high levels of Ser7 and Ser5 phosphorylation on the C-terminal repeat domain (CTD) of Pol II. Ser2 phosphorylation levels increase until 600-1,000 nucleotides downstream of the TSS and do not correlate with recruitment of Spt6 and Pcf11, which bind the Ser2-phosphorylated CTD in vitro. This indicates CTD-independent recruitment mechanisms and CTD masking in vivo. Elongation complexes are productive and disassemble in a two-step 3′ transition. Paf1, Spt16 (part of the FACT complex), and the CTD kinases Bur1 and Ctk1 exit upstream of the polyadenylation site, whereas Spt4, Spt5, Spt6, Spn1 (also called Iws1) and Elf1 exit downstream. Transitions are uniform and independent of gene length, type and expression.correlate with the in vivo occupancy of two factors that bind the phosphorylated CTD in vitro. General elongation complexes are active, as their gene occupancy predicts mRNA expression levels. RESULTS Genome-wide profiling reveals Pol II on a majority of genesWe determined genome-wide occupancy profiles by ChIP in exponentially growing Saccharomyces cerevisiae strains expressing tandem affinity purification (TAP)-tagged proteins (Online Methods). Chromatin immunoprecipitation was performed as described 11 , with modifications (Online Methods and Supplementary Methods). Enriched DNA fragments of an average size of 250 nucleotides (nt; Supplementary Fig. 1) were analyzed with tiling microarrays that cover the yeast genome at 4-nt resolution 12 . For data normalization, we developed a procedure that corrects for nonspecific antibody binding by using input measurements as well as mock immunoprecipitations (Supplementary Methods). Data from two or three highly reproducible replicates were averaged (Supplementary Table 1). The profile for the Pol II subunit Rpb3 (Fig. 1) matched previous profiles 13 obtained with different strains, experimental protocols and array platforms, but the new profile showed more details (Supplementary Fig. 2).Pol II was observed at genes encoding proteins, small nuclear RNA and small nucleolar RNA, and at regions producing cryptic unstable and unannotated transcripts 14 , but was lacking at genes transcribed by Pol I and Pol III (Fig. 1a and Supplementary Fig. 3). Of 4,366 yeast genes with annotated TSS and pA sites 15 , 2,465 (56%) showed Pol II peak occupancies above 20%, consistent with transcription of most of the Gene transcription begins with the assembly of Pol II and its initiation factors on promoter DNA. Pol II then starts mRNA synthesis and exchanges initiation factors for elongation factors, which are required for chromatin passage and RNA processing [1][2][3] . Whereas Pol II is unphosp...

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.

SummaryThe Nrd1-Nab3-Sen1 (NNS) complex is essential for controlling pervasive transcription and generating sn/snoRNAs in S. cerevisiae. The NNS complex terminates transcription of noncoding RNA genes and promotes exosome-dependent processing/degradation of the released transcripts. The Trf4-Air2-Mtr4 (TRAMP) complex polyadenylates NNS target RNAs and favors their degradation. NNS-dependent termination and degradation are coupled, but the mechanism underlying this coupling remains enigmatic. Here we provide structural and functional evidence demonstrating that the same domain of Nrd1p interacts with RNA polymerase II and Trf4p in a mutually exclusive manner, thus defining two alternative forms of the NNS complex, one involved in termination and the other in degradation. We show that the Nrd1-Trf4 interaction is required for optimal exosome activity in vivo and for the stimulation of polyadenylation of NNS targets by TRAMP in vitro. We propose that transcription termination and RNA degradation are coordinated by switching between two alternative partners of the NNS complex.