Sequence-Resolved Detection of Pausing by Single RNA Polymerase Molecules

Transcriptional pausing by RNA polymerase (RNAP) plays an important role in the regulation of gene expression. Defined, sequence-specific pause sites have been identified biochemically. Single-molecule studies have also shown that bacterial RNAP pauses frequently during transcriptional elongation, but the relationship of these "ubiquitous" pauses to the underlying DNA sequence has been uncertain. We employed an ultrastable optical-trapping assay to follow the motion of individual molecules of RNAP transcribing templates engineered with repeated sequences carrying imbedded, sequence-specific pause sites of known regulatory function. Both the known and ubiquitous pauses appeared at reproducible locations, identified with base-pair accuracy. Ubiquitous pauses were associated with DNA sequences that show similarities to regulatory pause sequences. Data obtained for the lifetimes and efficiencies of pauses support a model where the transition to pausing branches off of the normal elongation pathway and is mediated by a common elemental state, which corresponds to the ubiquitous pause.

[1]  Jeffrey W. Roberts,et al.  Function of E. coli RNA Polymerase σ Factor- σ70 in Promoter-Proximal Pausing , 1996, Cell.

[2]  W. Gu,et al.  Identification of a decay in transcription potential that results in elongation factor dependence of RNA polymerase II , 1995, The Journal of Biological Chemistry.

[3]  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.

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

[5]  C. Bustamante,et al.  Single-molecule study of transcriptional pausing and arrest by E. coli RNA polymerase. , 2000, Science.

[6]  P. V. von Hippel,et al.  The elongation-termination decision in transcription. , 1992, Science.

[7]  J. Roberts,et al.  Function of E. coli RNA polymerase sigma factor sigma 70 in promoter-proximal pausing. , 1996, Cell.

[8]  P. V. von Hippel,et al.  Active Escherichia coli transcription elongation complexes are functionally homogeneous. , 2002, Journal of molecular biology.

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

[10]  M. Chamberlin,et al.  Pausing and termination of transcription within the early region of bacteriophage T7 DNA in vitro. , 1981, The Journal of biological chemistry.

[11]  C. Yanofsky,et al.  Transcription attenuation. , 1988, The Journal of biological chemistry.

[12]  R. Landick,et al.  Roles of RNA:DNA hybrid stability, RNA structure, and active site conformation in pausing by human RNA polymerase II. , 2001, Journal of molecular biology.

[13]  C. Job,et al.  Abortive intermediates in transcription by wheat-germ RNA polymerase II. Dynamic aspects of enzyme/template interactions in selection of the enzyme synthetic mode. , 1990, The Biochemical journal.

[14]  D. Erie.  The many conformational states of RNA polymerase elongation complexes and their roles in the regulation of transcription. , 2002, Biochimica et biophysica acta.

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

[16]  Gary D. Stormo,et al.  Displaying the information contents of structural RNA alignments: the structure logos , 1997, Comput. Appl. Biosci..

[17]  N. Proudfoot,et al.  Specific transcriptional pausing activates polyadenylation in a coupled in vitro system. , 1999, Molecular cell.

[18]  J. Lis,et al.  Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS. , 2005, Molecular cell.

[19]  J. Clarke,et al.  Mechanical and chemical unfolding of a single protein: a comparison. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[20]  C. Chan,et al.  Dissection of the his leader pause site by base substitution reveals a multipartite signal that includes a pause RNA hairpin. , 1993, Journal of molecular biology.

[21]  R. Young,et al.  RNA polymerase II. , 1991, Annual review of biochemistry.

[22]  E. Nudler,et al.  Coupling between transcription termination and RNA polymerase inchworming , 1995, Cell.

[23]  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.

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

[25]  W. Greenleaf,et al.  Direct observation of base-pair stepping by RNA polymerase , 2005, Nature.

[26]  R. Landick,et al.  Allosteric Control of RNA Polymerase by a Site That Contacts Nascent RNA Hairpins , 2001, Science.

[27]  K. J. Harrington,et al.  Balanced branching in transcription termination , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[28]  R. G. Keene,et al.  Transcriptional pausing at +62 of the HIV-1 nascent RNA modulates formation of the TAR RNA structure. , 1998, Molecular cell.

[29]  M. Kashlev,et al.  Overextended RNA:DNA hybrid as a negative regulator of RNA polymerase II processivity. , 2000, Journal of molecular biology.

[30]  D. K. Hawley,et al.  Promoter Proximal Sequences Modulate RNA Polymerase II Elongation by a Novel Mechanism , 1996, Cell.

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

[32]  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.

[33]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[34]  R. Landick,et al.  Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

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

[36]  Robert Landick,et al.  Diversity in the Rates of Transcript Elongation by Single RNA Polymerase Molecules* , 2004, Journal of Biological Chemistry.

[37]  Michelle D. Wang,et al.  A single-molecule technique to study sequence-dependent transcription pausing. , 2004, Biophysical journal.

[38]  M. J. Bailey,et al.  RfaH and the ops element, components of a novel system controlling bacterial transcription elongation , 1997, Molecular microbiology.

[39]  Anirvan M. Sengupta,et al.  Thermodynamic and kinetic modeling of transcriptional pausing. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[40]  Joshua W. Shaevitz,et al.  Backtracking by single RNA polymerase molecules observed at near-base-pair resolution , 2003, Nature.

[41]  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.

[42]  Michelle D. Wang,et al.  Sequence-dependent kinetic model for transcription elongation by RNA polymerase. , 2004, Journal of molecular biology.

[43]  A. Kornblihtt,et al.  A slow RNA polymerase II affects alternative splicing in vivo. , 2003, Molecular cell.

[44]  K. Struhl,et al.  TFIIS Enhances Transcriptional Elongation through an Artificial Arrest Site In Vivo , 2001, Molecular and Cellular Biology.

[45]  P. V. von Hippel,et al.  Transcript elongation and termination are competitive kinetic processes. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[46]  Walter Gilbert,et al.  Starting and Stopping Sequences for the RNA Polymerase , 1976 .

[47]  P. V. von Hippel,et al.  Multiple RNA polymerase conformations and GreA: control of the fidelity of transcription. , 1993, Science.

[48]  Lakshmi V. Madabusi,et al.  Promoter-Proximal Pausing on the hsp70Promoter in Drosophila melanogaster Depends on the Upstream Regulator , 2000, Molecular and Cellular Biology.

[49]  C. Yanofsky,et al.  Regulation by transcription attenuation in bacteria: how RNA provides instructions for transcription termination/antitermination decisions. , 2002, BioEssays : news and reviews in molecular, cellular and developmental biology.

[50]  Vasily M Studitsky,et al.  Nature of the nucleosomal barrier to RNA polymerase II. , 2005, Molecular cell.