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.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Nature Communications Open Access 17 September 2021
Cellular and Molecular Life Sciences Open Access 19 June 2021
Cell Death & Disease Open Access 21 February 2020
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Pokholok, D.K., Hannett, N.M. & Young, R.A. Exchange of RNA polymerase II initiation and elongation factors during gene expression in vivo. Mol. Cell 9, 799–809 (2002).
Orphanides, G. & Reinberg, D. RNA polymerase II elongation through chromatin. Nature 407, 471–475 (2000).
Orphanides, G. & Reinberg, D. A unified theory of gene expression. Cell 108, 439–451 (2002).
Komarnitsky, P., Cho, E.-J. & Buratowski, S. Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. Genes Dev. 14, 2452–2460 (2000).
Schroeder, S.C., Schwer, B., Shuman, S. & Bentley, D. Dynamic association of capping enzymes with transcribing RNA polymerase II. Genes Dev. 14, 2435–2440 (2000).
Buratowski, S. Progression through the RNA polymerase II CTD cycle. Mol. Cell 36, 541–546 (2009).
Perales, R. & Bentley, D. “Cotranscriptionality”: the transcription elongation complex as a nexus for nuclear transactions. Mol. Cell 36, 178–191 (2009).
Meinhart, A., Kamenski, T., Hoeppner, S., Baumli, S. & Cramer, P. A structural perspective of CTD function. Genes Dev. 19, 1401–1415 (2005).
Hirose, Y. & Manley, J.L. RNA polymerase II and the integration of nuclear events. Genes Dev. 14, 1415–1429 (2000).
Venters, B.J. & Pugh, B.F. A canonical promoter organization of the transcription machinery and its regulators in the Saccharomyces genome. Genome Res. 19, 360–371 (2009).
Aparicio, O. et al. Chromatin immunoprecipitation for determining the association of proteins with specific genomic sequences in vivo. in Current Protocols in Molecular Biology (eds. Ausubel, F.A. et al.) Ch. 21, Unit 21.3 (Wiley, 2005).
David, L. et al. A high-resolution map of transcription in the yeast genome. Proc. Natl. Acad. Sci. USA 103, 5320–5325 (2006).
Jasiak, A.J. et al. Genome-associated RNA polymerase II includes the dissociable Rpb4/7 subcomplex. J. Biol. Chem. 283, 26423–26427 (2008).
Xu, Z. et al. Bidirectional promoters generate pervasive transcription in yeast. Nature 457, 1033–1037 (2009).
Nagalakshmi, U. et al. The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320, 1344–1349 (2008).
Dengl, S., Mayer, A., Sun, M. & Cramer, P. Structure and in vivo requirement of the yeast Spt6 SH2 domain. J. Mol. Biol. 389, 211–225 (2009).
Kuehner, J.N. & Brow, D.A. Quantitative analysis of in vivo initiator selection by yeast RNA polymerase II supports a scanning model. J. Biol. Chem. 281, 14119–14128 (2006).
Kostrewa, D. et al. RNA polymerase II-TFIIB structure and mechanism of transcription initiation. Nature 462, 323–330 (2009).
Renner, D.B., Yamaguchi, Y., Wada, T., Handa, H. & Price, D.H. A highly purified RNA polymerase II elongation control system. J. Biol. Chem. 276, 42601–42609 (2001).
Chapman, R.D. et al. Transcribing RNA polymerase II is phosphorylated at CTD residue serine-7. Science 318, 1780–1782 (2007).
Kim, M., Suh, H., Cho, E.-J. & Buratowski, S. Phosphorylation of the yeast Rpb1 C-terminal domain at serines 2, 5, and 7. J. Biol. Chem. 284, 26421–26426 (2009).
Akhtar, M.S. et al. TFIIH kinase places bivalent marks on the carboxy-terminal domain of RNA polymerase II. Mol. Cell 34, 387–393 (2009).
Boeing, S., Rigault, C., Heidemann, M., Eick, D. & Meisterernst, M. RNA polymerase II C-terminal heptarepeat domain Ser-7 phosphorylation is established in a Mediator-dependent fashion. J. Biol. Chem. 285, 188–196 (2010).
Murray, S., Udupa, R., Yao, S., Hartzog, G. & Prelich, G. Phosphorylation of the RNA polymerase II carboxy-terminal domain by the Bur1 cyclin-dependent kinase. Mol. Cell. Biol. 21, 4089–4096 (2001).
Liu, Y. et al. Phosphorylation of the transcription elongation factor Spt5 by yeast Bur1 kinase stimulates recruitment of the PAF complex. Mol. Cell. Biol. 29, 4852–4863 (2009).
Rodriguez, C.R. et al. Kin28, the TFIIH-Associated carboxy-terminal domain kinase, facilitates the recruitment of mRNA processing machinery to RNA polymerase II. Mol. Cell. Biol. 20, 104–112 (2000).
Yoh, S.M., Cho, H., Pickle, L., Evans, R.M. & Jones, K.A. The Spt6 SH2 domain binds Ser2-P RNAPII to direct Iws1-dependent mRNA splicing and export. Genes Dev. 21, 160–174 (2007).
Barillà, D., Lee, B.A. & Proudfoot, N.J. Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 98, 445–450 (2001).
Nechaev, S. et al. Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila. Science 327, 335–338 (2010).
Zeitlinger, J. et al. RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo. Nat. Genet. 39, 1512–1516 (2007).
Core, L.J., Waterfall, J.J. & Lis, J.T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008).
Rahl, P.B. et al. c-Myc regulates transcriptional pause release. Cell 141, 432–445 (2010).
Jiang, C. & Pugh, B.F. Nucleosome positioning and gene regulation: advances through genomics. Nat. Rev. Genet. 10, 161–172 (2009).
Radonjic, M. et al. Genome-wide analyses reveal RNA polymerase II located upstream of genes poised for rapid response upon S. cerevisiae stationary phase exit. Mol. Cell 18, 171–183 (2005).
Chen, H.-T., Warfield, L. & Hahn, S. The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex. Nat. Struct. Mol. Biol. 14, 696–703 (2007).
Hirtreiter, A. et al. Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif. Nucleic Acids Res. 38, 4040–4051 (2010).
Guo, M. et al. Core structure of the yeast Spt4-Spt5 complex: a conserved module for regulation of transcription elongation. Structure 16, 1649–1658 (2008).
Lindstrom, D.L. et al. Dual roles for Spt5 in pre-mRNA processing and transcription elongation revealed by identification of Spt5-associated proteins. Mol. Cell. Biol. 23, 1368–1378 (2003).
Krogan, N.J. et al. RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach. Mol. Cell. Biol. 22, 6979–6992 (2002).
Prather, D., Krogan, N.J., Emili, A., Greenblatt, J.F. & Winston, F. Identification and characterization of Elf1, a conserved transcription elongation factor in Saccharomyces cerevisiae. Mol. Cell. Biol. 25, 10122–10135 (2005).
Qiu, H., Hu, C. & Hinnebusch, A.G. Phosphorylation of the Pol II CTD by KIN28 enhances BUR1/BUR2 recruitment and Ser2 CTD phosphorylation near promoters. Mol. Cell 33, 752–762 (2009).
Zhou, K., Kuo, W.H.W., Fillingham, J. & Greenblatt, J.F. Control of transcriptional elongation and cotranscriptional histone modification by the yeast BUR kinase substrate Spt5. Proc. Natl. Acad. Sci. USA 106, 6956–6961 (2009).
Laribee, R.N. et al. BUR kinase selectively regulates H3 K4 trimethylation and H2B ubiquitylation through recruitment of the PAF elongation complex. Curr. Biol. 15, 1487–1493 (2005).
Stuwe, T. et al. The FACT Spt16 “peptidase” domain is a histone H3–H4 binding module. Proc. Natl. Acad. Sci. USA 105, 8884–8889 (2008).
Belotserkovskaya, R. et al. FACT facilitates transcription-dependent nucleosome alteration. Science 301, 1090–1093 (2003).
Ahn, S.H., Keogh, M.-C. & Buratowski, S. Ctk1 promotes dissociation of basal transcription factors from elongating RNA polymerase II. EMBO J. 28, 205–212 (2009).
Kim, M., Ahn, S.-H., Krogan, N.J., Greenblatt, J.F. & Buratowski, S. Transitions in RNA polymerase II elongation complexes at the 3′ ends of genes. EMBO J. 23, 354–364 (2004).
Keogh, M.-C., Podolny, V. & Buratowski, S. Bur1 kinase is required for efficient transcription elongation by RNA polymerase II. Mol. Cell. Biol. 23, 7005–7018 (2003).
Glover-Cutter, K., Kim, S., Espinosa, J. & Bentley, D.L. RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes. Nat. Struct. Mol. Biol. 15, 71–78 (2008).
Kaplan, C.D., Holland, M.J. & Winston, F. Interaction between transcription elongation factors and mRNA 3′-end formation at the Saccharomyces cerevisiae GAL10–GAL7 locus. J. Biol. Chem. 280, 913–922 (2005).
Kaplan, C.D., Laprade, L. & Winston, F. Transcription elongation factors repress transcription initiation from cryptic sites. Science 301, 1096–1099 (2003).
Ahn, S.H., Kim, M. & Buratowski, S. Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3′ end processing. Mol. Cell 13, 67–76 (2004).
Hesselberth, J.R. et al. Global mapping of protein-DNA interactions in vivo by digital genomic footprinting. Nat. Methods 6, 283–289 (2009).
Yuan, G.C. et al. Genome-scale identification of nucleosome positions in S. cerevisiae. Science 309, 626–630 (2005).
Fan, X., Lamarre-Vincent, N., Wang, Q. & Struhl, K. Extensive chromatin fragmentation improves enrichment of protein binding sites in chromatin immunoprecipitation experiments. Nucleic Acids Res. 36, e125 (2008).
We thank H. Feldmann and D. Martin for help, D. Eick (Helmholtz Zentrum München) for providing antibodies and A. Tresch for discussions. J.S. was supported by the Deutsche Forschungsgemeinschaft and SFB646. P.C. was supported by the Deutsche Forschungsgemeinschaft, the SFB646, the TR5, the Nanosystems Initiative Munich NIM, the Elitenetzwerk Bayern, the Jung-Stiftung and the Fonds der Chemischen Industrie.
The authors declare no competing financial interests.
About this article
Cite this article
Mayer, A., Lidschreiber, M., Siebert, M. et al. Uniform transitions of the general RNA polymerase II transcription complex. Nat Struct Mol Biol 17, 1272–1278 (2010). https://doi.org/10.1038/nsmb.1903
This article is cited by
Current Genetics (2022)
Nature Communications (2021)
Cellular and Molecular Life Sciences (2021)
Current Genetics (2021)