Unified two‐metal mechanism of RNA synthesis and degradation by RNA polymerase

In DNA‐dependent RNA polymerases, reactions of RNA synthesis and degradation are performed by the same active center (in contrast to DNA polymerases in which they are separate). We propose a unified catalytic mechanism for multisubunit RNA polymerases based on the analysis of its 3′–5′ exonuclease reaction in the context of crystal structure. The active center involves a symmetrical pair of Mg2+ ions that switch roles in synthesis and degradation. One ion is retained permanently and the other is recruited ad hoc for each act of catalysis. The weakly bound Mg2+ is stabilized in the active center in different modes depending on the type of reaction: during synthesis by the β,γ‐phosphates of the incoming substrate; and during hydrolysis by the phosphates of a non‐base‐paired nucleoside triphosphate. The latter mode defines a transient, non‐specific nucleoside triphosphate‐binding site adjacent to the active center, which may serve as a gateway for polymerization of substrates.

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

[2]  F. Werner,et al.  A recombinant RNA polymerase II-like enzyme capable of promoter-specific transcription. , 2002, Molecular cell.

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

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

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

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

[7]  S. Darst,et al.  A Structural Model of Transcription Elongation , 2000 .

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

[9]  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.

[10]  D. K. Hawley,et al.  Transcriptional Fidelity and Proofreading by RNA Polymerase II , 1998, Cell.

[11]  T. Steitz,et al.  Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates. , 1998, Journal of molecular biology.

[12]  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.

[13]  M. Kashlev,et al.  RNA Polymerase Switches between Inactivated and Activated States By Translocating Back and Forth along the DNA and the RNA* , 1997, The Journal of Biological Chemistry.

[14]  E. Nudler,et al.  The RNA–DNA Hybrid Maintains the Register of Transcription by Preventing Backtracking of RNA Polymerase , 1997, Cell.

[15]  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.

[16]  V. Markovtsov,et al.  Mapping of Catalytic Residues in the RNA Polymerase Active Center , 1996, Science.

[17]  E. Nudler,et al.  Histidine-tagged RNA polymerase of Escherichia coli and transcription in solid phase. , 1996, Methods in enzymology.

[18]  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.

[19]  M. Rudd,et al.  The active site of RNA polymerase II participates in transcript cleavage within arrested ternary complexes. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[20]  K. Severinov,et al.  A non-essential domain of Escherichia coli RNA polymerase required for the action of the termination factor Alc. , 1994, The Journal of biological chemistry.

[21]  K.,et al.  Hydrolytic cleavage of nascent RNA in RNA polymerase III ternary transcription complexes. , 1994, The Journal of biological chemistry.

[22]  S. Borukhov,et al.  Recombinant Escherichia coli RNA polymerase: purification of individually overexpressed subunits and in vitro assembly. , 1993, Protein expression and purification.

[23]  D. Luse,et al.  The increment of SII-facilitated transcript cleavage varies dramatically between elongation competent and incompetent RNA polymerase II ternary complexes. , 1993, The Journal of biological chemistry.

[24]  S. Borukhov,et al.  Transcript cleavage factors from E. coli , 1993, Cell.

[25]  D. K. Hawley,et al.  Identification of a 3'-->5' exonuclease activity associated with human RNA polymerase II. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[26]  S. Shuman,et al.  Nascent RNA cleavage by purified ternary complexes of vaccinia RNA polymerase. , 1993, The Journal of biological chemistry.

[27]  D. Reines.  Elongation factor-dependent transcript shortening by template-engaged RNA polymerase II. , 1992, The Journal of biological chemistry.

[28]  M. Kashlev,et al.  A beta subunit mutation disrupting the catalytic function of Escherichia coli RNA polymerase. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[29]  H. Dixon,et al.  The mechanism of pyrophosphorolysis of RNA by RNA polymerase. Endowment of RNA polymerase with artificial exonuclease activity. , 1984, The Biochemical journal.

[30]  W. R. McClure.  On the mechanism of streptolydigin inhibition of Escherichia coli RNA polymerase. , 1980, The Journal of biological chemistry.

[31]  V. Armstrong,et al.  Mechanistic studies on deoxyribonucleic acid dependent ribonucleic acid polymerase from Escherichia coli using phosphorothioate analogues. 2. The elongation reaction. , 1979, Biochemistry.

[32]  V. Armstrong,et al.  Mechanistic studies on deoxyribonucleic acid dependent ribonucleic acid polymerase from Escherichia coli using phosphorothioate analogues. 1. Initiation and pyrophosphate exchange reactions. , 1979, Biochemistry.

[33]  R. Koren,et al.  Magnetic resonance and kinetic studies of the role of the divalent cation activator of RNA polymerase from Escherichia coli. , 1977, Biochemistry.

[34]  D. Hoard,et al.  CONVERSION OF MONO- AND OLIGODEOXYRIBONUCLEOTIDES TO 5-TRIPHOSPHATES. , 1965, Journal of the American Chemical Society.

[35]  P. Cramer,et al.  Structural Basis of Transcription : RNA Polymerase II at 2 . 8 Å Resolution , 2022 .