NTP-driven Translocation by Human RNA Polymerase II*

We report a “running start, two-bond” protocol to analyze elongation by human RNA polymerase II (RNAP II). In this procedure, the running start allowed us to measure rapid rates of elongation and provided detailed insight into the RNAP II mechanism. Formation of two bonds was tracked to ensure that at least one translocation event was analyzed. By using this method, RNAP II is stalled briefly at a defined template position before restoring the next NTP. Significantly, slow reaction steps are identified both before and after phosphodiester bond synthesis, and both of these steps can be highly dependent on the next templated NTP. The initial and final NTP-driven events, however, are not identical, because the slow step after chemistry, which includes translocation and pyrophosphate release, is regulated differently by elongation factors hepatitis δ antigen and transcription factor IIF. Because recovery from a stall and the processive transition from one bond to the next can be highly NTP-dependent, we conclude that translocation can be driven by the incoming substrate NTP, a model fully consistent with the RNAP II elongation complex structure.

[1]  P. V. Hippel,et al.  An Integrated Model of the Transcription Complex in Elongation, Termination, and Editing , 1998 .

[2]  R. Conaway,et al.  Roles for both the RAP30 and RAP74 subunits of transcription factor IIF in transcription initiation and elongation by RNA polymerase II. , 1994, The Journal of biological chemistry.

[3]  M. Lai,et al.  Rolling Circle Replication of Hepatitis Delta Virus RNA Is Carried Out by Two Different Cellular RNA Polymerases , 2002, Journal of Virology.

[4]  K. Johnson,et al.  Conformational coupling in DNA polymerase fidelity. , 1993, Annual review of biochemistry.

[5]  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.

[6]  Kenneth A. Johnson,et al.  1 Transient-State Kinetic Analysis of Enzyme Reaction Pathways , 1992 .

[7]  Michelle D. Wang,et al.  Single molecule analysis of RNA polymerase elongation reveals uniform kinetic behavior , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[8]  M. Tsai,et al.  Use of 2-aminopurine and tryptophan fluorescence as probes in kinetic analyses of DNA polymerase beta. , 2002, Biochemistry.

[9]  K. Johnson,et al.  Rapid quench kinetic analysis of polymerases, adenosinetriphosphatases, and enzyme intermediates. , 1995, Methods in enzymology.

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

[11]  F. Jülicher,et al.  Motion of RNA polymerase along DNA: a stochastic model. , 1998, Biophysical journal.

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

[13]  P. Kuzmič,et al.  Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. , 1996, Analytical biochemistry.

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

[15]  D. Luse,et al.  Factor-stimulated RNA polymerase II transcribes at physiological elongation rates on naked DNA but very poorly on chromatin templates. , 1992, The Journal of biological chemistry.

[16]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[17]  H. Handa,et al.  Assay of transient state kinetics of RNA polymerase II elongation. , 2003, Methods in enzymology.

[18]  T. Wada,et al.  The regulation of elongation by eukaryotic RNA polymerase II: a recent view. , 2001, Molecules and cells.

[19]  A. Dvir,et al.  Promoter escape by RNA polymerase II. , 2002, Biochimica et biophysica acta.

[20]  M. Dreyfus,et al.  NTP concentration effects on initial transcription by T7 RNAP indicate that translocation occurs through passive sliding and reveal that divergent promoters have distinct NTP concentration requirements for productive initiation. , 1998, Journal of molecular biology.

[21]  H. Handa,et al.  A Highly Purified RNA Polymerase II Elongation Control System* , 2001, The Journal of Biological Chemistry.

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

[23]  Michelle D. Wang,et al.  Force and velocity measured for single molecules of RNA polymerase. , 1998, Science.

[24]  T. Elston,et al.  Force generation in RNA polymerase. , 1998, Biophysical journal.

[25]  H. Handa,et al.  HIV and hepatitis delta virus: evolution takes different paths to relieve blocks in transcriptional elongation. , 2002, Microbes and infection.

[26]  Steven M. Block,et al.  Transcription Against an Applied Force , 1995, Science.

[27]  Z. Burton,et al.  Importance of codon preference for production of human RAP74 and reconstitution of the RAP30/74 complex. , 1994, Protein expression and purification.

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

[29]  Jinhong Chang,et al.  In vivo RNA‐directed transcription, with template switching, by a mammalian RNA polymerase , 2002, The EMBO journal.

[30]  宁北芳,et al.  疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A , 2005 .

[31]  M. Guthold,et al.  Single‐Molecule Study Reveals a Complex E. coli RNA Polymerase , 2001, Chembiochem : a European journal of chemical biology.

[32]  G. Cagney,et al.  RNA Polymerase II Elongation Factors of Saccharomyces cerevisiae: a Targeted Proteomics Approach , 2002, Molecular and Cellular Biology.

[33]  J. Taylor,et al.  The RNAs of hepatitis delta virus are copied by RNA polymerase II in nuclear homogenates , 1993, Journal of virology.

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

[35]  E. Nudler,et al.  Mechanistic model of the elongation complex of Escherichia coli RNA polymerase. , 1998, Cold Spring Harbor symposia on quantitative biology.

[36]  G. Waksman,et al.  Crystal structures of a ddATP‐, ddTTP‐, ddCTP, and ddGTP‐ trapped ternary complex of Klentaq1: Insights into nucleotide incorporation and selectivity , 2001, Protein science : a publication of the Protein Society.

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

[38]  Ming-Daw Tsai,et al.  A reexamination of the nucleotide incorporation fidelity of DNA polymerases. , 2002, Biochemistry.

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

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

[41]  D. Reinberg,et al.  Role of the mammalian transcription factors IIF, IIS, and IIX during elongation by RNA polymerase II , 1991, Molecular and Cellular Biology.

[42]  A. Sluder,et al.  Dynamic interaction between a Drosophila transcription factor and RNA polymerase II , 1989, Molecular and cellular biology.

[43]  Z. Burton,et al.  The RAP74 Subunit of Human Transcription Factor IIF Has Similar Roles in Initiation and Elongation , 1999, Molecular and Cellular Biology.

[44]  Ali Shilatifard,et al.  Factors regulating the transcriptional elongation activity of RNA polymerase II , 1998, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[45]  R. Conaway,et al.  Dual Roles for Transcription Factor IIF in Promoter Escape by RNA Polymerase II* , 1999, The Journal of Biological Chemistry.

[46]  Y. Nedialkov,et al.  A Key Role for the α1 Helix of Human RAP74 in the Initiation and Elongation of RNA Chains* , 2002, The Journal of Biological Chemistry.

[47]  Nancy R. Forde,et al.  Using mechanical force to probe the mechanism of pausing and arrest during continuous elongation by Escherichia coli RNA polymerase , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[48]  C. F. Kostrub,et al.  Production of human RAP30 and RAP74 in bacterial cells. , 1993, Protein expression and purification.

[49]  K. Johnson,et al.  An induced-fit kinetic mechanism for DNA replication fidelity: direct measurement by single-turnover kinetics. , 1991, Biochemistry.

[50]  K. Yano,et al.  Stimulation of RNA Polymerase II Elongation by Hepatitis Delta Antigen , 2001, Science.

[51]  M. Lai,et al.  RNA-Dependent Replication and Transcription of Hepatitis Delta Virus RNA Involve Distinct Cellular RNA Polymerases , 2000, Molecular and Cellular Biology.

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

[53]  A. Shilatifard,et al.  Control of elongation by RNA polymerase II. , 2000, Trends in biochemical sciences.

[54]  J. Liu,et al.  Insight into the catalytic mechanism of DNA polymerase beta: structures of intermediate complexes. , 2001, Biochemistry.

[55]  P. Sharp,et al.  A high-efficiency HeLa cell nuclear transcription extract. , 1988, DNA.