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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...

Summary Individual mammalian cells exhibit large variability in cellular volume even with the same absolute DNA content and so must compensate for differences in DNA concentration in order to maintain constant concentration of gene expression products. Using single molecule counting and computational image analysis, we show that transcript abundance correlates with cellular volume at the single cell level due to increased global transcription in larger cells. Cell fusion experiments establish that increased cellular content itself can directly increase transcription. Quantitative analysis shows that this mechanism measures the ratio of cellular volume to DNA content, mostly likely through sequestration of a transcriptional factor to DNA. Analysis of transcriptional bursts reveals a separate mechanism for gene dosage compensation after DNA replication that enables proper transcriptional output during early and late S-phase. Our results provide a framework for quantitatively understanding the relationships between DNA content, cell size and gene expression variability in single cells.

Rates of mRNA synthesis and decay can be measured on a genome-wide scale in yeast by dynamic transcriptome analysis (DTA), which combines non-perturbing metabolic RNA labeling with dynamic kinetic modeling.DTA reveals that most mRNA synthesis rates are around several transcripts per cell and cell cycle, and most mRNA half-lives range around a median of 11 min.DTA realistically monitors the cellular response to osmotic stress with higher sensitivity and temporal resolution than transcriptomics, and can be used to follow changes in RNA metabolism in gene regulatory systems.