Role of the RNA polymerase trigger loop in catalysis and pausing

The trigger loop (TL) is a polymorphous component of RNA polymerase (RNAP) that makes direct substrate contacts and promotes nucleotide addition when folded into an α-helical hairpin (trigger helices, TH). However, the roles of the TL/TH in transcript cleavage, catalysis, substrate selectivity and pausing remain ill defined. Based on in vitro assays of Escherichia coli RNAP bearing specific TL/TH alterations, we report that neither intrinsic nor regulator-assisted transcript cleavage of backtracked RNA requires formation of the TH. We find that the principal contribution of TH formation to rapid nucleotidyl transfer is steric alignment of the reactants rather than acid-base catalysis, and that the TL/TH cannot be the sole contributor to substrate selectivity. The similar effects of TL/TH substitutions on pausing and nucleotide addition provide additional support for the view that TH formation is rate-limiting for escape from nonbacktracked pauses.

[1]  P. Cramer,et al.  Structural basis of transcription inhibition by α-amanitin and implications for RNA polymerase II translocation , 2008, Nature Structural &Molecular Biology.

[2]  A. Das,et al.  Intrinsic transcript cleavage activity of RNA polymerase. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[3]  M. Chamberlin,et al.  Ribonucleic acid chain elongation by Escherichia coli ribonucleic acid polymerase. I. Isolation of ternary complexes and the kinetics of elongation. , 1974, The Journal of biological chemistry.

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

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

[6]  Robert Landick,et al.  The flap domain is required for pause RNA hairpin inhibition of catalysis by RNA polymerase and can modulate intrinsic termination. , 2003, Molecular cell.

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

[8]  J. Arnold,et al.  Nucleic acid polymerases employ a general acid for nucleotidyl transfer , 2008, Nature Structural &Molecular Biology.

[9]  William J. Rice,et al.  Structure and Function of the Transcription Elongation Factor GreB Bound to Bacterial RNA Polymerase , 2003, Cell.

[10]  C. Chan,et al.  Discontinuous movements of DNA and RNA in RNA polymerase accompany formation of a paused transcription complex , 1995, Cell.

[11]  T. Steitz,et al.  Structural biology: A mechanism for all polymerases , 1998, Nature.

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

[13]  M. Kozlov,et al.  Donation of catalytic residues to RNA polymerase active center by transcription factor Gre , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Simone C. Wiesler,et al.  Bridge helix and trigger loop perturbations generate superactive RNA polymerases , 2008, Journal of biology.

[15]  S. Yokoyama,et al.  Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution , 2002, Nature.

[16]  C. Chan,et al.  Multiple interactions stabilize a single paused transcription intermediate in which hairpin to 3' end spacing distinguishes pause and termination pathways. , 1997, Journal of molecular biology.

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

[18]  J. Arnold,et al.  Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases , 2007, Proceedings of the National Academy of Sciences.

[19]  B. Shafer,et al.  Mutations in the Saccharomyces cerevisiae RPB1 Gene Conferring Hypersensitivity to 6-Azauracil , 2006, Genetics.

[20]  E. Koonin,et al.  Evolution of bacterial RNA polymerase: implications for large-scale bacterial phylogeny, domain accretion, and horizontal gene transfer. , 2004, Gene.

[21]  M. Kashlev,et al.  Crucial role of the RNA:DNA hybrid in the processivity of transcription. , 1998, Molecules and Cells.

[22]  K. Murakami,et al.  Co-overexpression of Escherichia coliRNA Polymerase Subunits Allows Isolation and Analysis of Mutant Enzymes Lacking Lineage-specific Sequence Insertions* , 2003, The Journal of Biological Chemistry.

[23]  Jeffrey W. Roberts,et al.  Mechanism of intrinsic transcription termination and antitermination. , 1999, Science.

[24]  D. Vassylyev,et al.  Discrimination against Deoxyribonucleotide Substrates by Bacterial RNA Polymerase* , 2004, Journal of Biological Chemistry.

[25]  R. Landick,et al.  Direct Versus Limited-step Reconstitution Reveals Key Features of an RNA Hairpin-stabilized Paused Transcription Complex* , 2007, Journal of Biological Chemistry.

[26]  Robert Landick,et al.  Transcriptional pausing without backtracking , 2009, Proceedings of the National Academy of Sciences.

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

[28]  R. Landick.  Active-Site Dynamics in RNA Polymerases , 2004, Cell.

[29]  J. Liu,et al.  DNA polymerase beta: contributions of template-positioning and dNTP triphosphate-binding residues to catalysis and fidelity. , 2000, Biochemistry.

[30]  K. Murakami,et al.  Structure and function of lineage-specific sequence insertions in the bacterial RNA polymerase beta' subunit. , 2005, Journal of molecular biology.

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

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

[33]  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.

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

[35]  J. Richardson.  Loading Rho to Terminate Transcription , 2003, Cell.

[36]  Elio A. Abbondanzieri,et al.  Ubiquitous Transcriptional Pausing Is Independent of RNA Polymerase Backtracking , 2003, Cell.

[37]  V. Markovtsov,et al.  Modular organization of the catalytic center of RNA polymerase. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

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

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

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

[41]  M. Kashlev,et al.  Mechanism of sequence-specific pausing of bacterial RNA polymerase , 2009, Proceedings of the National Academy of Sciences.

[42]  Yulia Yuzenkova,et al.  Transcript-Assisted Transcriptional Proofreading , 2006, Science.

[43]  Arkady Mustaev,et al.  Unified two‐metal mechanism of RNA synthesis and degradation by RNA polymerase , 2003, The EMBO journal.

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