Templated nucleoside triphosphate binding to a noncatalytic site on RNA polymerase regulates transcription

The regulation of RNA synthesis by RNA polymerase (RNAP) is essential for proper gene expression. Crystal structures of RNAP reveal two channels: the main channel that contains the downstream DNA and a secondary channel that leads directly to the catalytic site. Although nucleoside triphosphates (NTPs) have been seen only in the catalytic site and the secondary channel in these structures, several models of transcription elongation, based on biochemical studies, propose that template-dependent binding of NTPs in the main channel regulates RNA synthesis. These models, however, remain controversial. We used transient state kinetics and a mutant of RNAP to investigate the role of the main channel in regulating nucleotide incorporation. Our data indicate that a NTP specific for the i + 2 template position can bind to a noncatalytic site and increase the rate of RNA synthesis and that the NTP bound to this site can be shuttled directly into the catalytic site. We also identify fork loop 2, which lies across from the downstream DNA, as a functional component of this site. Taken together, our data support the existence of a noncatalytic template-specific NTP binding site in the main channel that is involved in the regulation of nucleotide incorporation. NTP binding to this site could promote high-fidelity processive synthesis under a variety of environmental conditions and allow DNA sequence-mediated regulatory signals to be communicated to the active site.

[1]  P. Cramer,et al.  Mechanism of transcriptional stalling at cisplatin-damaged DNA , 2007, Nature Structural &Molecular Biology.

[2]  J. Strathern,et al.  Rpb9 Subunit Controls Transcription Fidelity by Delaying NTP Sequestration in RNA Polymerase II*♦ , 2009, The Journal of Biological Chemistry.

[3]  Michelle D. Wang,et al.  Sequence-dependent kinetic model for transcription elongation by RNA polymerase. , 2004, Journal of molecular biology.

[4]  R. Ebright,et al.  Structures of RNA polymerase-antibiotic complexes. , 2009, Current opinion in structural biology.

[5]  M. Kashlev,et al.  Millisecond phase kinetic analysis of elongation catalyzed by human, yeast, and Escherichia coli RNA polymerase. , 2009, Methods.

[6]  M. Levitt,et al.  Structural Basis of Transcription: Backtracked RNA Polymerase II at 3.4 Angstrom Resolution , 2009, Science.

[7]  Craig D. Kaplan,et al.  The RNA polymerase II trigger loop functions in substrate selection and is directly targeted by alpha-amanitin. , 2008, Molecular cell.

[8]  D. Erie,et al.  Downstream DNA Sequence Effects on Transcription Elongation , 2003, Journal of Biological Chemistry.

[9]  Zachary F. Burton,et al.  Human RNA Polymerase II Elongation in Slow Motion: Role of the TFIIF RAP74 α1 Helix in Nucleoside Triphosphate-Driven Translocation , 2005, Molecular and Cellular Biology.

[10]  R. Landick,et al.  Downstream DNA selectively affects a paused conformation of human RNA polymerase II. , 2004, Journal of molecular biology.

[11]  M. Chamberlin,et al.  Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[12]  M. Levitt,et al.  Diffusion of nucleoside triphosphates and role of the entry site to the RNA polymerase II active center. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[13]  J. Register,et al.  Rapid kinetic analysis of transcription elongation by Escherichia coli RNA polymerase. , 2008, Journal of molecular biology.

[14]  Steven M. Block,et al.  Sequence-Resolved Detection of Pausing by Single RNA Polymerase Molecules , 2006, Cell.

[15]  Michael Feig,et al.  NTP-driven translocation and regulation of downstream template opening by multi-subunit RNA polymerases. , 2005, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[16]  P. Cramer,et al.  Template misalignment in multisubunit RNA polymerases and transcription fidelity. , 2006, Molecules and Cells.

[17]  P. V. von Hippel,et al.  The elongation-termination decision in transcription. , 1992, Science.

[18]  P. Cramer,et al.  Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA. , 2009, Molecular cell.

[19]  Tahir H. Tahirov,et al.  Structural basis for transcription elongation by bacterial RNA polymerase , 2007, Nature.

[20]  S. Benkovic,et al.  Kinetic mechanism of DNA polymerase I (Klenow fragment): identification of a second conformational change and evaluation of the internal equilibrium constant. , 1991, Biochemistry.

[21]  P. V. von Hippel,et al.  Transcript elongation and termination are competitive kinetic processes. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[22]  D. Temiakov,et al.  A mechanism of nucleotide misincorporation during transcription due to template-strand misalignment. , 2006, Molecular cell.

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

[24]  P. V. von Hippel,et al.  Multiple RNA polymerase conformations and GreA: control of the fidelity of transcription. , 1993, Science.

[25]  Jonathan Tennyson,et al.  Water vapour in the atmosphere of a transiting extrasolar planet , 2007, Nature.

[26]  Smita S. Patel,et al.  Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. , 1991, Biochemistry.

[27]  R. Landick,et al.  Transcription pausing by Escherichia coli RNA polymerase is modulated by downstream DNA sequences. , 1990, The Journal of biological chemistry.

[28]  Julio O. Ortiz,et al.  A movie of the RNA polymerase nucleotide addition cycle. , 2009, Current opinion in structural biology.

[29]  T. Tahirov,et al.  Structural basis of transcription inhibition by antibiotic streptolydigin. , 2005, Molecular cell.

[30]  C. Chan,et al.  The Salmonella typhimurium his operon leader region contains an RNA hairpin-dependent transcription pause site. Mechanistic implications of the effect on pausing of altered RNA hairpins. , 1989, The Journal of biological chemistry.

[31]  S. Borukhov,et al.  GreA protein: a transcription elongation factor from Escherichia coli. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[32]  D. Erie,et al.  Allosteric Binding of Nucleoside Triphosphates to RNA Polymerase Regulates Transcription Elongation , 2001, Cell.

[33]  Scott R Kennedy,et al.  Forks, pincers, and triggers: the tools for nucleotide incorporation and translocation in multi-subunit RNA polymerases. , 2009, Current opinion in structural biology.

[34]  Robert Landick,et al.  A central role of the RNA polymerase trigger loop in active-site rearrangement during transcriptional pausing. , 2007, Molecular cell.

[35]  Michael Feig,et al.  Dynamic error correction and regulation of downstream bubble opening by human RNA polymerase II. , 2005, Molecular cell.

[36]  Irina Artsimovitch,et al.  Structural basis for substrate loading in bacterial RNA polymerase , 2007, Nature.

[37]  J. Arnold,et al.  Poliovirus RNA-dependent RNA polymerase (3Dpol): pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of Mg2+. , 2004, Biochemistry.

[38]  D. Erie.  The many conformational states of RNA polymerase elongation complexes and their roles in the regulation of transcription. , 2002, Biochimica et biophysica acta.

[39]  D. Bushnell,et al.  Structural Basis of Transcription Nucleotide Selection by Rotation in the RNA Polymerase II Active Center , 2004, Cell.

[40]  Zachary F Burton,et al.  Translocation by multi-subunit RNA polymerases. , 2010, Biochimica et biophysica acta.

[41]  Zachary F Burton,et al.  A Tunable Ratchet Driving Human RNA Polymerase II Translocation Adjusted by Accurately Templated Nucleoside Triphosphates Loaded at Downstream Sites and by Elongation Factors* , 2007, Journal of Biological Chemistry.

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

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

[44]  Arkady Mustaev,et al.  A Ratchet Mechanism of Transcription Elongation and Its Control , 2005, Cell.

[45]  A. D. Clark,et al.  Inhibition of Bacterial RNA Polymerase by Streptolydigin: Stabilization of a Straight-Bridge-Helix Active-Center Conformation , 2005, Cell.

[46]  R. Burgess,et al.  A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. , 1975, Biochemistry.

[47]  Michelle D. Wang,et al.  Molecular mechanism of transcription inhibition by peptide antibiotic Microcin J25. , 2004, Molecular cell.

[48]  K. Severinov,et al.  Crystal Structure of Thermus aquaticus Core RNA Polymerase at 3.3 Å Resolution , 1999, Cell.

[49]  R. Landick,et al.  Roles of RNA:DNA hybrid stability, RNA structure, and active site conformation in pausing by human RNA polymerase II. , 2001, Journal of molecular biology.

[50]  M. Chamberlin,et al.  Parameters affecting transcription termination by Escherichia coli RNA polymerase. I. Analysis of 13 rho-independent terminators. , 1992, Journal of molecular biology.

[51]  Hiroshi Handa,et al.  NTP-driven Translocation by Human RNA Polymerase II* , 2003, The Journal of Biological Chemistry.

[52]  J. Arnold,et al.  Poliovirus RNA-dependent RNA polymerase (3Dpol): pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of Mn2+. , 2004, Biochemistry.

[53]  R. Landick,et al.  Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[54]  Jennifer L. Knight,et al.  Antibacterial peptide microcin J25 inhibits transcription by binding within and obstructing the RNA polymerase secondary channel. , 2004, Molecular cell.

[55]  J. Strathern,et al.  Transient reversal of RNA polymerase II active site closing controls fidelity of transcription elongation. , 2008, Molecular cell.