Structural basis for transcription inhibition by tagetitoxin

Tagetitoxin (Tgt) inhibits transcription by an unknown mechanism. A structure at a resolution of 2.4 Å of the Thermus thermophilus RNA polymerase (RNAP)–Tgt complex revealed that the Tgt-binding site within the RNAP secondary channel overlaps that of the stringent control effector ppGpp, which partially protects RNAP from Tgt inhibition. Tgt binding is mediated exclusively through polar interactions with the β and β′ residues whose substitutions confer resistance to Tgt in vitro. Importantly, a Tgt phosphate, together with two active site acidic residues, coordinates the third Mg2+ ion, which is distinct from the two catalytic metal ions. We show that Tgt inhibits all RNAP catalytic reactions and propose a mechanism in which the Tgt-bound Mg2+ ion has a key role in stabilization of an inactive transcription intermediate. Remodeling of the active site by metal ions could be a common theme in the regulation of catalysis by nucleic acid enzymes.

[1]  S. Nechaev,et al.  Mutations of Bacterial RNA Polymerase Leading to Resistance to Microcin J25* , 2002, The Journal of Biological Chemistry.

[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]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[4]  Patrick Cramer,et al.  Structure and function of RNA polymerase II. , 2004, Advances in protein chemistry.

[5]  R M Esnouf,et al.  Further additions to MolScript version 1.4, including reading and contouring of electron-density maps. , 1999, Acta crystallographica. Section D, Biological crystallography.

[6]  M. Kashlev,et al.  Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3' end of the RNA intact and extruded. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Naohiro Matsugaki,et al.  Allosteric Modulation of the RNA Polymerase Catalytic Reaction Is an Essential Component of Transcription Control by Rifamycins , 2005, Cell.

[8]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[9]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

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

[11]  D. Jin,et al.  The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like "stringent" RNA polymerases in Escherichia coli. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

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

[13]  John W. Foster,et al.  DksA A Critical Component of the Transcription Initiation Machinery that Potentiates the Regulation of rRNA Promoters by ppGpp and the Initiating NTP , 2004, Cell.

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

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

[16]  Thomas A Steitz,et al.  The Structural Mechanism of Translocation and Helicase Activity in T7 RNA Polymerase , 2004, Cell.

[17]  C. Gross,et al.  Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance. , 1988, Journal of molecular biology.

[18]  O. Laptenko,et al.  Transcript cleavage factors GreA and GreB act as transient catalytic components of RNA polymerase , 2003, The EMBO journal.

[19]  R. Burgess,et al.  Tagetitoxin inhibition of RNA polymerase III transcription results from enhanced pausing at discrete sites and is template-dependent. , 1992, Journal of Biological Chemistry.

[20]  R. Durbin,et al.  Tagetitoxin inhibits RNA synthesis directed by RNA polymerases from chloroplasts and Escherichia coli. , 1990, The Journal of biological chemistry.

[21]  R. Landick,et al.  The Transcriptional Regulator RfaH Stimulates RNA Chain Synthesis after Recruitment to Elongation Complexes by the Exposed Nontemplate DNA Strand , 2002, Cell.

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

[23]  S. Yokoyama,et al.  Structural Basis for Transcription Regulation by Alarmone ppGpp , 2004, Cell.

[24]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

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

[26]  Shigeyuki Yokoyama,et al.  Regulation through the Secondary Channel—Structural Framework for ppGpp-DksA Synergism during Transcription , 2004, Cell.

[27]  P. Kraulis A program to produce both detailed and schematic plots of protein structures , 1991 .

[28]  Wei Yang,et al.  Crystal Structures of RNase H Bound to an RNA/DNA Hybrid: Substrate Specificity and Metal-Dependent Catalysis , 2005, Cell.

[29]  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.

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

[31]  P. Cramer,et al.  The dynamic machinery of mRNA elongation. , 2005, Current opinion in structural biology.

[32]  R. Durbin,et al.  Tagetitoxin affects plastid development in seedling leaves of wheat , 1985, Planta.

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

[34]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

[35]  Shigeyuki Yokoyama,et al.  Structural Basis for Substrate Selection by T7 RNA Polymerase , 2004, Cell.

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

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

[38]  R. Durbin,et al.  Mechanistic aspects of tagetitoxin inhibition of RNA polymerase from Escherichia coli. , 1994, Biochemistry.

[39]  R. Burgess,et al.  Tagetitoxin: a new inhibitor of eukaryotic transcription by RNA polymerase III. , 1990, The Journal of biological chemistry.

[40]  Clement S. Chu,et al.  A New Class of Bacterial RNA Polymerase Inhibitor Affects Nucleotide Addition , 2003, Science.

[41]  P. Cramer,et al.  Architecture of the RNA Polymerase II-TFIIS Complex and Implications for mRNA Cleavage , 2003, Cell.

[42]  R. Burgess,et al.  RNA Polymerases from Bacillus subtilisand Escherichia coli Differ in Recognition of Regulatory Signals In Vitro , 2000, Journal of bacteriology.

[43]  Samuel H. Wilson,et al.  Critical role of magnesium ions in DNA polymerase beta's closing and active site assembly. , 2004, Journal of the American Chemical Society.

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