Structural Basis for Substrate Selection by T7 RNA Polymerase

The mechanism by which nucleotide polymerases select the correct substrate is of fundamental importance to the fidelity of DNA replication and transcription. During the nucleotide addition cycle, pol I DNA polymerases undergo the transition from a catalytically inactive "open" to an active "closed" conformation. All known determinants of substrate selection are associated with the "closed" state. To elucidate if this mechanism is conserved in homologous single subunit RNA polymerases (RNAPs), we have determined the structure of T7 RNAP elongation complex with the incoming substrate analog. Surprisingly, the substrate specifically binds to RNAP in the "open" conformation, where it is base paired with the acceptor template base, while Tyr639 provides discrimination of ribose versus deoxyribose substrates. The structure therefore suggests a novel mechanism, in which the substrate selection occurs prior to the isomerization to the catalytically active conformation. Modeling of multisubunit RNAPs suggests that this mechanism might be universal for all RNAPs.

[1]  W. Mcallister.  Transcription by T7 RNA Polymerase , 1997 .

[2]  Thomas A. Steitz,et al.  Structural Basis for the Transition from Initiation to Elongation Transcription in T7 RNA Polymerase , 2002, Science.

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

[4]  S. Darst,et al.  Bacterial RNA polymerase. , 2001, Current opinion in structural biology.

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

[6]  R. Sousa,et al.  A mutant T7 RNA polymerase as a DNA polymerase. , 1995, The EMBO journal.

[7]  Christophe Benoist,et al.  Structural Basis for the Transition from Initiation to Elongation Transcription in T 7 RNA Polymerase , 2022 .

[8]  T. Kunkel,et al.  Side Chains That Influence Fidelity at the Polymerase Active Site of Escherichia coli DNA Polymerase I (Klenow Fragment)* , 1999, The Journal of Biological Chemistry.

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

[10]  Yong Je Chung,et al.  Crystal structure of bacteriophage T7 RNA polymerase at 3.3 Å resolution , 1993, Nature.

[11]  C. M. Joyce,et al.  Choosing the right sugar: how polymerases select a nucleotide substrate. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[12]  C. M. Joyce,et al.  A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

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

[14]  A. Woody,et al.  Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity. , 1992, Journal of molecular biology.

[15]  Sean J. Johnson,et al.  Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Gabriel Waksman,et al.  Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation , 1998, The EMBO journal.

[17]  James R. Kiefer,et al.  Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal , 1998, Nature.

[18]  W. Mcallister,et al.  The phage RNA polymerases are related to DNA polymerases and reverse transcriptases , 1993, Molecular microbiology.

[19]  S. Kochetkov,et al.  Structural–Functional Analysis of Bacteriophage T7 RNA Polymerase , 2002, Biochemistry (Moscow).

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

[21]  S. Doublié,et al.  The mechanism of action of T7 DNA polymerase. , 1998, Current opinion in structural biology.

[22]  L. Loeb,et al.  In vivo mutagenesis by Escherichia coli DNA polymerase I. Ile(709) in motif A functions in base selection. , 2001, The Journal of biological chemistry.

[23]  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.

[24]  M. Anikin,et al.  Characterization of T7 RNA Polymerase Transcription Complexes Assembled on Nucleic Acid Scaffolds* , 2002, The Journal of Biological Chemistry.

[25]  R. Sousa,et al.  T7 RNA polymerase elongation complex structure and movement. , 2000, Journal of molecular biology.

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

[27]  S. Doublié,et al.  Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution , 1998, Nature.

[28]  M. Anikin,et al.  Major Conformational Changes Occur during the Transition from an Initiation Complex to an Elongation Complex by T7 RNA Polymerase* , 2002, The Journal of Biological Chemistry.

[29]  M. Saraste,et al.  FEBS Lett , 2000 .

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

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

[32]  L. Brieba,et al.  Roles of histidine 784 and tyrosine 639 in ribose discrimination by T7 RNA polymerase. , 2000, Biochemistry.

[33]  S. Yokoyama,et al.  Crystallization and preliminary crystallographic analysis of T7 RNA polymerase elongation complex. , 2003, Acta crystallographica. Section D, Biological crystallography.

[34]  T. Kunkel,et al.  Base Miscoding and Strand Misalignment Errors by Mutator Klenow Polymerases with Amino Acid Substitutions at Tyrosine 766 in the O Helix of the Fingers Subdomain* , 1997, The Journal of Biological Chemistry.

[35]  S. Yokoyama,et al.  Structure of a T7 RNA polymerase elongation complex at 2.9 Å resolution , 2002, Nature.

[36]  T. Kunkel,et al.  Discrimination against purine–pyrimidine mispairs in the polymerase active site of DNA polymerase I: A structural explanation , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[37]  R. Sousa,et al.  Characterization of a set of T7 RNA polymerase active site mutants. , 1994, The Journal of biological chemistry.

[38]  G. Waksman,et al.  Crystal structures of the Klenow fragment of Thermus aquaticus DNA polymerase I complexed with deoxyribonucleoside triphosphates , 1998, Protein science : a publication of the Protein Society.

[39]  Yufei Huang,et al.  Determinants of ribose specificity in RNA polymerization: effects of Mn2+ and deoxynucleoside monophosphate incorporation into transcripts. , 1997, Biochemistry.

[40]  H. Lipson.  Crystal Structures , 1949, Nature.

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

[42]  J. Steitz,et al.  A general two-metal-ion mechanism for catalytic RNA. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

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

[44]  R. Sousa,et al.  A model for the mechanism of polymerase translocation. , 1997, Journal of molecular biology.

[45]  L. Brieba,et al.  Misincorporation by wild-type and mutant T7 RNA polymerases: identification of interactions that reduce misincorporation rates by stabilizing the catalytically incompetent open conformation. , 2000, Biochemistry.

[46]  T. Steitz,et al.  Structure of T7 RNA polymerase complexed to the transcriptional inhibitor T7 lysozyme , 1998, The EMBO journal.

[47]  B. Chernov,et al.  Mutants of T7 RNA polymerase that are able to synthesize both RNA and DNA , 1995, FEBS letters.

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

[49]  K. Murakami,et al.  Bacterial RNA polymerases: the wholo story. , 2003, Current opinion in structural biology.

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

[51]  L. Loeb,et al.  Prokaryotic DNA polymerase I: evolution, structure, and "base flipping" mechanism for nucleotide selection. , 2001, Journal of molecular biology.

[52]  T. Steitz,et al.  A unified polymerase mechanism for nonhomologous DNA and RNA polymerases. , 1994, Science.

[53]  Pre-steady-state and steady-state kinetic studies on transcription initiation catalyzed by T7 RNA polymerase and its active-site mutants K631R and Y639F. , 1998, Biochemistry.

[54]  D. Patra,et al.  Mutations in T7 RNA polymerase that support the proposal for a common polymerase active site structure. , 1992, The EMBO journal.

[55]  Yufei Huang,et al.  Mechanism of ribose 2'-group discrimination by an RNA polymerase. , 1997, Biochemistry.

[56]  V. Markovtsov,et al.  Swing-gate model of nucleotide entry into the RNA polymerase active center. , 2002, Molecular cell.

[57]  Ping Zhou,et al.  Crystal structures of [18]aneN6H2K[Co(CN)6].4H2O, [16]aneN4H2K[Co(CN)6] and [12]aneN4H3[Co(CN)6].2H2O. Insight into the electrostatic and hydrogen-bonding interaction in self-assembling supercomplexes , 1999 .

[58]  S. Yoshida,et al.  O-helix Mutant T664P of Thermus aquaticus DNA Polymerase I , 2001, The Journal of Biological Chemistry.

[59]  W. Mcallister,et al.  The specificity loop of T7 RNA polymerase interacts first with the promoter and then with the elongating transcript, suggesting a mechanism for promoter clearance. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[60]  T. Littlejohn,et al.  Swiss-PDB Viewer (Deep View). , 2001, Briefings in bioinformatics.

[61]  T. Steitz,et al.  Structure of a transcribing T7 RNA polymerase initiation complex. , 1999, Science.