Transcription regulation occurs frequently through promoter-associated pausing of RNA polymerase II (Pol II). We developed a Precision nuclear Run-On and sequencing assay (PRO-seq) to map the genome-wide distribution of transcriptionally-engaged Pol II at base-pair resolution. Pol II accumulates immediately downstream of promoters, at intron-exon junctions that are efficiently used for splicing, and over 3' poly-adenylation sites. Focused analyses of promoters reveal that pausing is not fixed relative to initiation sites nor is it specified directly by the position of a particular core promoter element or the first nucleosome. Core promoter elements function beyond initiation, and when optimally positioned they act collectively to dictate the position and strength of pausing. We test this ‘Complex Interaction’ model with insertional mutagenesis of the Drosophila Hsp70 core promoter.

In the eukaryotic genome, the thousands of genes that encode messenger RNA are transcribed by a molecular machine called RNA polymerase II. Analysing the distribution and status of RNA polymerase II across a genome has provided crucial insights into the long-standing mysteries of transcription and its regulation. These studies identify points in the transcription cycle where RNA polymerase II accumulates after encountering a rate-limiting step. When coupled with genome-wide mapping of transcription factors, these approaches identify key regulatory steps and factors and, importantly, provide an understanding of the mechanistic generalities, as well as the rich diversities, of gene regulation.The genetic information encoded in the DNA of eukaryotic genes is transcribed into RNA by large molecular machines called RNA polymerases. One of these machines, RNA polymerase II (Pol II), transcribes all the protein-coding genes. The control of Pol II activity is highly modulated at individual genes, and this specific regulation is critical for both the homeostasis of cells and the programmed development of multicellular organisms. The execution of this regulation is dictated by combinatorial molecular interactions of transcription factors with each other and with specific DNA sequences at each gene. Modern biochemical and molecular methods coupled with genetics and genomics have identified thousands of factors that participate in regulated transcription 1 . Most of these factors are proteins, but a growing number of them are RNAs. They enable Pol II to gain access to the gene's promoter, to initiate RNA synthesis at the transcription start site (TSS) of the gene and to generate a productively elongating transcription complex that produces a full-length RNA transcript.The thousands of transcription factors involved in the transcription process may be true regulatory factors or simply critical cogs in the cycle of transcription. True regulatory factors are likely to represent only a fraction of the total number of factors that are important for gene expression. As an analogy, consider a motor vehicle: a car has numerous crucial components and processes that are required to achieve acceleration and proper speed (cylinders, spark plugs, tyres and so on), but components regulated by the driver are limited to the ignition, the steering wheel, the accelerator and brake pedals, and the gear stick. Therefore, it is important to identify the true regulatory factors and the associated biochemical processes that execute gene regulation. The status and local density of the ultimate target of regulation, the transcription machine Pol II, have proved extremely useful in assessing the steps in the transcription cycle that are rate limiting and are altered in vivo by particular transcription factors (the driver in the above analogy).

Drosophila contains one (dCDK12) and humans contain two (hCDK12 and hCDK13) proteins that are the closest evolutionary relatives of yeast Ctk1, the catalytic subunit of the major elongation-phase C-terminal repeat domain (CTD) kinase in Saccharomyces cerevisiae, CTDK-I. However, until now, neither CDK12 nor CDK13 has been demonstrated to be a bona fide CTD kinase. Using Drosophila, we demonstrate that dCDK12 (CG7597) is a transcription-associated CTD kinase, the ortholog of yCtk1. Fluorescence microscopy reveals that the distribution of dCDK12 on formaldehyde-fixed polytene chromosomes is virtually identical to that of hyperphosphorylated RNA polymerase II (RNAPII), but is distinct from that of P-TEFb (dCDK9 + dCyclin T). Chromatin immunoprecipitation (ChIP) experiments confirm that dCDK12 is present on the transcribed regions of active Drosophila genes. Compared with P-TEFb, dCDK12 amounts are lower at the 59 end and higher in the middle and at the 39 end of genes (both normalized to RNAPII). Appropriately, Drosophila dCDK12 purified from nuclear extracts manifests CTD kinase activity in vitro. Intriguingly, we find that cyclin K is associated with purified dCDK12, implicating it as the cyclin subunit of this CTD kinase. Most importantly, we demonstrate that RNAi knockdown of dCDK12 in S2 cells alters the phosphorylation state of the CTD, reducing its Ser2 phosphorylation levels. Similarly, in human HeLa cells, we show that hCDK13 purified from nuclear extracts displays CTD kinase activity in vitro, as anticipated. Also, we find that chimeric (yeast/human) versions of Ctk1 containing the kinase homology domains of hCDK12/13 (or hCDK9) are functional in yeast cells (and also in vitro); using this system, we show that a bur1 ts mutant is rescued more efficiently by a hCDK9 chimera than by a hCDK13 chimera, suggesting the following orthology relationships: Bur1 4 CDK9 and Ctk1 4 CDK12/13. Finally, we show that siRNA knockdown of hCDK12 in HeLa cells results in alterations in the CTD phosphorylation state. Our findings demonstrate that metazoan CDK12 and CDK13 are CTD kinases, and that CDK12 is orthologous to yeast Ctk1. The C-terminal repeat domain (CTD) of RNA polymerase II (RNAPII) is an intrinsically unstructured extension of the enzyme's largest subunit, RPB1. The CTD is composed of a tandem array of seven amino acid repeats with the consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 ; the number of heptad repeats varies from organism to organism and appears to correlate with genomic complexity (Corden 1990). Although dispensable for polymerase activity in vitro, the CTD is conserved throughout evolution and is essential to life (Zehring et al. 1988). The CTD's function is to coordinate transcription with nuclear processes such as mRNA processing and chromatin modification; it fulfills this role by serving as a selective binding scaffold for nuclear factors (for review, see Phatnani and Greenleaf 2006

During M phase, Endosulfine (Endos) family proteins are phosphorylated by Greatwall kinase (Gwl), and the resultant pEndos inhibits the phosphatase PP2A-B55, which would otherwise prematurely reverse many CDK-driven phosphorylations. We show here that PP2A-B55 is the enzyme responsible for dephosphorylating pEndos during M phase exit. The kinetic parameters for PP2A-B55’s action on pEndos are orders of magnitude lower than those for CDK-phosphorylated substrates, suggesting a simple model for PP2A-B55 regulation that we call inhibition by unfair competition. As the name suggests, during M phase PP2A-B55’s attention is diverted to pEndos, which binds much more avidly and is dephosphorylated more slowly than other substrates. When Gwl is inactivated during the M phase-to-interphase transition, the dynamic balance changes: pEndos dephosphorylated by PP2A-B55 cannot be replaced, so the phosphatase can refocus its attention on CDK-phosphorylated substrates. This mechanism explains simultaneously how PP2A-B55 and Gwl together regulate pEndos, and how pEndos controls PP2A-B55.DOI: http://dx.doi.org/10.7554/eLife.01695.001

Several eukaryotic transcription factors have been shown to modulate the elongation rate of RNA polymerase II (Pol II) on naked or chromatin-reconstituted templates in vitro. However, none of the tested factors have been shown to directly affect the elongation rate of Pol II in vivo. We performed a directed RNAi knock-down (KD) screen targeting 141 candidate transcription factors and identified multiple factors, including Spt6, that alter the induced Hsp70 transcript levels in Drosophila S2 cells. Spt6 is known to interact with both nucleosome structure and Pol II, and it has properties consistent with having a role in elongation. Here, ChIP assays of the first wave of Pol II after heat shock in S2 cells show that KD of Spt6 reduces the rate of Pol II elongation. Also, fluorescence recovery after photobleaching assays of GFP-Pol II in salivary gland cells show that this Spt6-dependent effect on elongation rate persists during steady-state-induced transcription, reducing the elongation rate from approximately 1100 to 500 bp/min. Furthermore, RNAi depletion of Spt6 reveals its broad requirement during different stages of development.