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Promoter Independent Abortive Transcription Assays Unravel Functional Interactions Between TFIIB and RNA Polymerase

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Gene Regulation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 977))

Abstract

TFIIB-like general transcription factors are required for transcription initiation by all eukaryotic and archaeal RNA polymerases (RNAPs). TFIIB facilitates both recruitment and post-recruitment steps of initiation; in particular, TFIIB stimulates abortive initiation. X-ray crystallography of TFIIB-RNAP II complexes shows that the TFIIB linker region penetrates the RNAP active center, yet the impact of this arrangement on RNAP activity and underlying mechanisms remains elusive. Promoter-independent abortive initiation assays exploit the intrinsic ability of RNAP enzymes to initiate transcription from nicked DNA templates and record the formation of the first phosphodiester bonds. These assays can be used to measure the effect of transcription factors such as TFIIB and RNAP mutations on abortive transcription.

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References

  1. Borukhov S, Nudler E (2008) RNA polymerase: the vehicle of transcription. Trends Microbiol 16:126–134

    Article  PubMed  CAS  Google Scholar 

  2. Buratowski S, Hahn S, Guarente L, Sharp PA (1989) Five intermediate complexes in transcription initiation by RNA polymerase II. Cell 56:549–561

    Article  PubMed  CAS  Google Scholar 

  3. Buratowski S, Sopta M, Greenblatt J, Sharp PA (1991) RNA polymerase II-associated proteins are required for a DNA conformation change in the transcription initiation complex. Proc Natl Acad Sci USA 88:7509–7513

    Article  PubMed  CAS  Google Scholar 

  4. Reese JC (2003) Basal transcription factors. Curr Opin Genet Dev 13:114–118

    Article  PubMed  CAS  Google Scholar 

  5. McClure WR (1985) Mechanism and control of transcription initiation in prokaryotes. Annu Rev Biochem 54:171–204

    Article  PubMed  CAS  Google Scholar 

  6. Walter G, Zillig W, Palm P, Fuchs E (1967) Initiation of DNA-dependent RNA synthesis and the effect of heparin on RNA polymerase. Eur J Biochem 3:194–201

    Article  PubMed  CAS  Google Scholar 

  7. Chen BS, Hampsey M (2004) Functional interaction between TFIIB and the Rpb2 subunit of RNA polymerase II: implications for the mechanism of transcription initiation. Mol Cell Biol 24:3983–3991

    Article  PubMed  CAS  Google Scholar 

  8. Artsimovitch I, Vassylyev DG (2006) Is it easy to stop RNA polymerase? Cell Cycle 5:399–404

    Article  PubMed  CAS  Google Scholar 

  9. Carpousis AJ, Gralla JD (1980) Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter. Biochemistry 19:3245–3253

    Article  PubMed  CAS  Google Scholar 

  10. Cheetham GM, Jeruzalmi D, Steitz TA (1999) Structural basis for initiation of transcription from an RNA polymerase-promoter complex. Nature 399:80–83

    Article  PubMed  CAS  Google Scholar 

  11. Gnatt AL, Cramer P, Fu J, Bushnell DA, Kornberg RD (2001) Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution. Science 292:1876–1882

    Article  PubMed  CAS  Google Scholar 

  12. Kapanidis AN, Margeat E, Ho SO, Kortkhonjia E, Weiss S, Ebright RH (2006) Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314:1144–1147

    Article  PubMed  Google Scholar 

  13. Krummel B, Chamberlin MJ (1989) RNA chain initiation by Escherichia coli RNA polymerase. Structural transitions of the enzyme in early ternary complexes. Biochemistry 28:7829–7842

    Article  PubMed  CAS  Google Scholar 

  14. Revyakin A, Liu C, Ebright RH, Strick TR (2006) Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science 314:1139–1143

    Article  PubMed  CAS  Google Scholar 

  15. Orphanides G, Reinberg D (2002) A unified theory of gene expression. Cell 108:439–451

    Article  PubMed  CAS  Google Scholar 

  16. Ebright RH (2000) RNA polymerase: structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II. J Mol Biol 304:687–698

    Article  PubMed  CAS  Google Scholar 

  17. Cheung AC, Sainsbury S, Cramer P (2011) Structural basis of initial RNA polymerase II transcription. EMBO J 30(23):4755–4763

    Article  PubMed  CAS  Google Scholar 

  18. Kostrewa D, Zeller ME, Armache KJ, Seizl M, Leike K, Thomm M, Cramer P (2009) RNA polymerase II-TFIIB structure and mechanism of transcription initiation. Nature 462(7271):323–330

    Article  PubMed  CAS  Google Scholar 

  19. Bushnell DA, Westover KD, Davis RE, Kornberg RD (2004) Structural basis of transcription: an RNA polymerase II-TFIIB cocrystal at 4.5 Angstroms. Science 303:983–988

    Article  PubMed  CAS  Google Scholar 

  20. Liu X, Bushnell DA, Wang D, Calero G, Kornberg RD (2010) Structure of an RNA Polymerase II-TFIIB Complex and the Transcription Initiation Mechanism. Science 327(5962):206–209

    Article  PubMed  CAS  Google Scholar 

  21. Chen HT, Hahn S (2004) Mapping the location of TFIIB within the RNA polymerase II transcription preinitiation complex: a model for the structure of the PIC. Cell 119:169–180

    Article  PubMed  CAS  Google Scholar 

  22. Chen HT, Hahn S (2003) Binding of TFIIB to RNA polymerase II: mapping the binding site for the TFIIB zinc ribbon domain within the preinitiation complex. Mol Cell 12:437–447

    Article  PubMed  CAS  Google Scholar 

  23. Wang D, Bushnell DA, Westover KD, Kaplan CD, Kornberg RD (2006) Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis. Cell 127:941–954

    Article  PubMed  CAS  Google Scholar 

  24. Boeger H, Bushnell DA, Davis R, Griesenbeck J, Lorch Y, Strattan JS, Westover KD, Kornberg RD (2005) Structural basis of eukaryotic gene transcription. FEBS Lett 579:899–903

    Article  PubMed  CAS  Google Scholar 

  25. Westover KD, Bushnell DA, Kornberg RD (2004) Structural basis of transcription: nucleotide selection by rotation in the RNA polymerase II active center. Cell 119:481–489

    Article  PubMed  CAS  Google Scholar 

  26. Westover KD, Bushnell DA, Kornberg RD (2004) Structural basis of transcription: separation of RNA from DNA by RNA polymerase II. Science 303:1014–1016

    Article  PubMed  CAS  Google Scholar 

  27. Bushnell DA, Cramer P, Kornberg RD (2002) Structural basis of transcription: alpha-amanitin-RNA polymerase II cocrystal at 2.8 A resolution. Proc Natl Acad Sci USA 99:1218–1222

    Article  PubMed  CAS  Google Scholar 

  28. Cramer P, Bushnell DA, Kornberg RD (2001) Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution. Science 292:1863–1876

    Article  PubMed  CAS  Google Scholar 

  29. Bell SD, Magill CP, Jackson SP (2001) Basal and regulated transcription in Archaea. Biochem Soc Trans 29:392–395

    Article  PubMed  CAS  Google Scholar 

  30. Parvin JD, Sharp PA (1993) DNA topology and a minimal set of basal factors for transcription by RNA polymerase II. Cell 73:533–540

    Article  PubMed  CAS  Google Scholar 

  31. Tyree CM, George CP, Lira-DeVito LM, Wampler SL, Dahmus ME, Zawel L, Kadonaga JT (1993) Identification of a minimal set of proteins that is sufficient for accurate initiation of transcription by RNA polymerase II. Genes Dev 7:1254–1265

    Article  PubMed  CAS  Google Scholar 

  32. Naidu S, Friedrich JK, Russell J, Zomerdijk JC (2011) TAF1B is a TFIIB-like component of the basal transcription machinery for RNA polymerase I. Science 333:1640–1642

    Article  PubMed  CAS  Google Scholar 

  33. Kassavetis GA, Geiduschek EP (2006) Transcription factor TFIIIB and transcription by RNA polymerase III. Biochem Soc Trans 34:1082–1087

    Article  PubMed  CAS  Google Scholar 

  34. Schramm L, Hernandez N (2002) Recruitment of RNA polymerase III to its target promoters. Genes Dev 16:2593–2620

    Article  PubMed  CAS  Google Scholar 

  35. Weinzierl RO, Wiesler SC (2011) Revealing the functions of TFIIB. Transcription 2: 254–257

    Google Scholar 

  36. Knutson BA, Hahn S (2011) Yeast Rrn7 and human TAF1B are TFIIB-related RNA polymerase I general transcription factors. Science 333:1637–1640

    Article  PubMed  CAS  Google Scholar 

  37. Buratowski S, Zhou H (1993) Functional domains of transcription factor TFIIB. Proc Natl Acad Sci USA 90:5633–5637

    Article  PubMed  CAS  Google Scholar 

  38. Werner F, Weinzierl RO (2005) Direct modulation of RNA polymerase core functions by basal transcription factors. Mol Cell Biol 25:8344–8355

    Article  PubMed  CAS  Google Scholar 

  39. Wiesler SC, Weinzierl RO (2011) The linker domain of basal transcription factor TFIIB controls distinct recruitment and transcription stimulation functions. Nucleic Acids Res 39:464–474

    Article  PubMed  CAS  Google Scholar 

  40. Werner F, Weinzierl RO (2002) A recombinant RNA polymerase II-like enzyme capable of promoter-specific transcription. Mol Cell 10:635–646

    Article  PubMed  CAS  Google Scholar 

  41. Tan L, Wiesler S, Trzaska D, Carney HC, Weinzierl RO (2008) Bridge helix and trigger loop perturbations generate superactive RNA polymerases. J Biol 7:40

    Article  PubMed  Google Scholar 

  42. Weinzierl RO (2010) The nucleotide addition cycle of RNA polymerase is controlled by two molecular hinges in the Bridge Helix domain. BMC Biol 8:134

    Article  PubMed  Google Scholar 

  43. Aposhian HV, Kornberg A (1962) Enzymatic synthesis of deoxyribonucleic acid. IX. The polymerase formed after T2 bacteriophage infection of Escherichia coli: a new enzyme. J Biol Chem 237:519–525

    PubMed  CAS  Google Scholar 

  44. Sun Y, Jacobson KB, Golovlev V (2007) A multienzyme bioluminescent time-resolved pyrophosphate assay. Anal Biochem 367:201–209

    Article  PubMed  CAS  Google Scholar 

  45. Ronaghi M, Karamohamed S, Pettersson B, Uhlen M, Nyren P (1996) Real-time DNA sequencing using detection of pyrophosphate release. Anal Biochem 242:84–89

    Article  PubMed  CAS  Google Scholar 

  46. Nyren P, Karamohamed S, Ronaghi M (1997) Detection of single-base changes using a bioluminometric primer extension assay. Anal Biochem 244:367–373

    Article  PubMed  CAS  Google Scholar 

  47. Karamohamed S, Nyren P (1999) Real-time detection and quantification of adenosine triphosphate sulfurylase activity by a bioluminometric approach. Anal Biochem 271:81–85

    Article  PubMed  CAS  Google Scholar 

  48. Lahser FC, Malcolm BA (2004) A continuous nonradioactive assay for RNA-dependent RNA polymerase activity. Anal Biochem 325:247–254

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by a Wellcome Project Grant (078043/Z/05/Z) to R.O.J.W.

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Authors and Affiliations

  1. Department of Life Sciences, Imperial College London, London, UK

    Simone C. Wiesler & Robert O. J. Weinzierl

  2. RNAP laboratory, Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, UK

    Finn Werner

Authors
  1. Simone C. Wiesler
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  2. Finn Werner
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  3. Robert O. J. Weinzierl
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Editors and Affiliations

  1. , Department of Chemistry, Purdue University, Oval Drive 560, West Lafayette, 47907-2084, Indiana, USA

    Minou Bina

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Wiesler, S.C., Werner, F., Weinzierl, R.O.J. (2013). Promoter Independent Abortive Transcription Assays Unravel Functional Interactions Between TFIIB and RNA Polymerase. In: Bina, M. (eds) Gene Regulation. Methods in Molecular Biology, vol 977. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-284-1_17

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  • DOI: https://doi.org/10.1007/978-1-62703-284-1_17

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-283-4

  • Online ISBN: 978-1-62703-284-1

  • eBook Packages: Springer Protocols

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