Dynamic architecture of a minimal RNA polymerase II open promoter complex.

The open promoter complex (OC) is a central intermediate during transcription initiation that contains a DNA bubble. Here, we employ single-molecule Förster resonance energy transfer experiments and Nano-Positioning System analysis to determine the three-dimensional architecture of a minimal OC consisting of promoter DNA, including a TATA box and an 11-nucleotide mismatched region around the transcription start site, TATA box-binding protein (TBP), RNA polymerase (Pol) II, and general transcription factor (TF)IIB and TFIIF. In this minimal OC, TATA-DNA and TBP reside above the Pol II cleft between clamp and protrusion domains. Downstream DNA is dynamically loaded into and unloaded from the Pol II cleft at a timescale of seconds. The TFIIB core domain is displaced from the Pol II wall, where it is located in the closed promoter complex. These results reveal large overall structural changes during the initiation-elongation transition, which are apparently accommodated by the intrinsic flexibility of TFIIB.

[1]  J. Gralla,et al.  Polymerase II promoter activation: closed complex formation and ATP-driven start site opening. , 1992, Science.

[2]  P. Cramer,et al.  RNA polymerase II–TFIIB structure and mechanism of transcription initiation , 2009, Nature.

[3]  Steven Hahn,et al.  Structure and mechanism of the RNA polymerase II transcription machinery , 2004, Nature Structural &Molecular Biology.

[4]  Jennifer L. Knight,et al.  Structural Organization of Bacterial RNA Polymerase Holoenzyme and the RNA Polymerase-Promoter Open Complex , 2002, Cell.

[5]  S. Hahn,et al.  Architecture of the Yeast RNA Polymerase II Open Complex and Regulation of Activity by TFIIF , 2011, Molecular and Cellular Biology.

[6]  Anton Meinhart,et al.  Structures of Complete RNA Polymerase II and Its Subcomplex, Rpb4/7* , 2005, Journal of Biological Chemistry.

[7]  P. Sharp,et al.  Five intermediate complexes in transcription initiation by RNA polymerase II , 1989, Cell.

[8]  P. Sigler,et al.  Structural basis of preinitiation complex assembly on human Pol II promoters , 2000, The EMBO journal.

[9]  J. Lis,et al.  DNA melting on yeast RNA polymerase II promoters. , 1993, Science.

[10]  K. Murakami,et al.  RNA polymerase and transcription elongation factor Spt4/5 complex structure , 2010, Proceedings of the National Academy of Sciences.

[11]  P. Cramer,et al.  Mediator head subcomplex Med11/22 contains a common helix bundle building block with a specific function in transcription initiation complex stabilization , 2011, Nucleic acids research.

[12]  S. Burley,et al.  Crystal structure of a TFIIB–TBP–TATA-element ternary complex , 1995, Nature.

[13]  K. Severinov,et al.  Structure‐based analysis of RNA polymerase function: the largest subunit's rudder contributes critically to elongation complex stability and is not involved in the maintenance of RNA–DNA hybrid length , 2002, The EMBO journal.

[14]  D. F. Ogletree,et al.  Probing the interaction between single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor , 1996, Summaries of Papers Presented at the Quantum Electronics and Laser Science Conference.

[15]  S. Hahn,et al.  A DNA-tethered cleavage probe reveals the path for promoter DNA in the yeast preinitiation complex , 2006, Nature Structural &Molecular Biology.

[16]  C. Walsh,et al.  Site-specific protein labeling by Sfp phosphopantetheinyl transferase , 2006, Nature Protocols.

[17]  E. Cho,et al.  Evidence That Transcription Factor IIB Is Required for a Post-assembly Step in Transcription Initiation* , 1999, The Journal of Biological Chemistry.

[18]  P. Cramer,et al.  Architecture of the RNA polymerase II–TFIIF complex revealed by cross-linking and mass spectrometry , 2010, EMBO Journal.

[19]  P. Cramer,et al.  Structural Basis of Transcription: An RNA Polymerase II Elongation Complex at 3.3 Å Resolution , 2001, Science.

[20]  C. Joo,et al.  Advances in single-molecule fluorescence methods for molecular biology. , 2008, Annual review of biochemistry.

[21]  Craig D. Kaplan,et al.  Structural Basis of Transcription: Role of the Trigger Loop in Substrate Specificity and Catalysis , 2006, Cell.

[22]  D. Bushnell,et al.  Structure of an RNA Polymerase II–TFIIB Complex and the Transcription Initiation Mechanism , 2010, Science.

[23]  R. Roeder,et al.  The role of general initiation factors in transcription by RNA polymerase II. , 1996, Trends in biochemical sciences.

[24]  P. Cramer,et al.  Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS. , 2004, Molecular cell.

[25]  Jennifer L. Knight,et al.  Distance-restrained docking of rifampicin and rifamycin SV to RNA polymerase using systematic FRET measurements: developing benchmarks of model quality and reliability. , 2005, Biophysical journal.

[26]  P B Sigler,et al.  The 2.1-A crystal structure of an archaeal preinitiation complex: TATA-box-binding protein/transcription factor (II)B core/TATA-box. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[27]  J. Greenblatt,et al.  Initiation of transcription by RNA polymerase II is limited by melting of the promoter DNA in the region immediately upstream of the initiation site. , 1994, The Journal of biological chemistry.

[28]  Helmut Grubmüller,et al.  Single-molecule FRET measures bends and kinks in DNA , 2008, Proceedings of the National Academy of Sciences.

[29]  Patrick Cramer,et al.  Architecture of the RNA polymerase–Spt4/5 complex and basis of universal transcription processivity , 2011, The EMBO journal.

[30]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[31]  T. Pardee,et al.  The N-terminal Region of Yeast TFIIB Contains Two Adjacent Functional Domains Involved in Stable RNA Polymerase II Binding and Transcription Start Site Selection* , 1998, The Journal of Biological Chemistry.

[32]  J. Michaelis,et al.  The Initiation Factor TFE and the Elongation Factor Spt4/5 Compete for the RNAP Clamp during Transcription Initiation and Elongation , 2011, Molecular cell.

[33]  S. Hahn,et al.  The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex , 2007, Nature Structural &Molecular Biology.

[34]  Mahadeb Pal,et al.  The role of the transcription bubble and TFIIB in promoter clearance by RNA polymerase II. , 2005, Molecular cell.

[35]  D. Bushnell,et al.  Structural basis of eukaryotic gene transcription , 2005, FEBS letters.

[36]  M Ikura,et al.  Human general transcription factor TFIIB: conformational variability and interaction with VP16 activation domain. , 1998, Biochemistry.

[37]  D. Luse,et al.  Transcription factor TFIIF is not required for initiation by RNA polymerase II, but it is essential to stabilize transcription factor TFIIB in early elongation complexes , 2011, Proceedings of the National Academy of Sciences.

[38]  Roger D Kornberg,et al.  Structural Basis of Transcription: An RNA Polymerase II-TFIIB Cocrystal at 4.5 Angstroms , 2004, Science.

[39]  Jens Michaelis,et al.  Application of the nano-positioning system to the analysis of fluorescence resonance energy transfer networks. , 2011, The journal of physical chemistry. B.

[40]  Jens Michaelis,et al.  Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex , 2009, Nucleic acids research.

[41]  Jens Michaelis,et al.  A nano-positioning system for macromolecular structural analysis , 2008, Nature Methods.

[42]  Jens Michaelis,et al.  Single-molecule tracking of mRNA exiting from RNA polymerase II , 2008, Proceedings of the National Academy of Sciences.

[43]  S. Hahn,et al.  Mapping the Location of TFIIB within the RNA Polymerase II Transcription Preinitiation Complex A Model for the Structure of the PIC , 2004, Cell.

[44]  P. Cramer,et al.  Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution , 2001, Science.

[45]  H. Grubmüller,et al.  Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1 , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[46]  P. Cramer,et al.  Structure–function analysis of the RNA polymerase cleft loops elucidates initial transcription, DNA unwinding and RNA displacement , 2007, Nucleic acids research.

[47]  S. McKinney,et al.  Analysis of single-molecule FRET trajectories using hidden Markov modeling. , 2006, Biophysical journal.

[48]  J. Ranish,et al.  Intermediates in formation and activity of the RNA polymerase II preinitiation complex: holoenzyme recruitment and a postrecruitment role for the TATA box and TFIIB. , 1999, Genes & development.

[49]  Taekjip Ha,et al.  DNA-binding orientation and domain conformation of the E. coli rep helicase monomer bound to a partial duplex junction: single-molecule studies of fluorescently labeled enzymes. , 2004, Journal of molecular biology.

[50]  T. Ha,et al.  Opening–closing dynamics of the mitochondrial transcription pre-initiation complex , 2011, Nucleic acids research.

[51]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[52]  T. Strick,et al.  Behavior of supercoiled DNA. , 1998, Biophysical journal.

[53]  J. Greenblatt,et al.  Recombinant TBP, transcription factor IIB, and RAP30 are sufficient for promoter recognition by mammalian RNA polymerase II. , 1992, The Journal of biological chemistry.

[54]  H R Drew,et al.  Structure of a B-DNA dodecamer: conformation and dynamics. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[55]  S. Hahn,et al.  Binding of TFIIB to RNA polymerase II: Mapping the binding site for the TFIIB zinc ribbon domain within the preinitiation complex. , 2003, Molecular cell.

[56]  D. Reinberg,et al.  Mechanism of ATP-dependent promoter melting by transcription factor IIH. , 2000, Science.