Nucleic Acid Enzymes and Nucleic Acids Studied at the Molecular Level

Summary

Principal Investigator: Steven M Block
Abstract: DESCRIPTION (provided by applicant): The Central Dogma of biology-whereby genetic information in DNA is replicated, transcribed, and ultimately translated into protein-is carried out by a handful of essential biomolecules that include polymerases, helicases, isomerases, and ribosomes. Together, these enzymes constitute a core group of sophisticated, bio- molecular machines. Understanding their function holds a key to understanding life, and by extension, to the treatment of human disease. Hard-won knowledge about biomolecules is informing work at the forefront of nanoscience, which hopes to harness tiny manmade devices to better the human condition. Put simply, we need to know how Nature's own machines work if we9re ever to fix them or to emulate them. A property shared by many nucleic acid-based enzymes is that they function as motors: once bound to DNA or RNA, they undergo repeated enzymatic cycles, often traveling considerable distances. This processive motion is ac- companied by force production and requires chemical energy. In contrast to classic mechanoenzymes like myosin, the properties of nucleic acid-based motors are modulated by an ever-changing template underfoot, yielding rich behavior. Although structural data are available for many such enzymes, comparatively little is known about their fundamental mechanisms. Recently, biophysical studies have been revolutionized by the ability to measure force and displacement at the level of individual molecules, using a variety of new techniques that include optical traps, nanometry, and fluorescence. Single-molecule techniques can supply critical information hitherto inaccessible to traditional approaches. In the previous grant cycle, my group succeeded in developing high-resolution optical trapping instrumentation that is able to register displacements down to the atomic level (~1E). Consequently, we can record from single bacterial RNA polymerase (RNAP) molecules as these step from base to base along DNA. Base-pair resolution makes it possible to sequence DNA based on enzyme motion, and points to new directions in nanoscience. Improved instrumental stability also allows us to reconstruct energy landscapes for folding transitions in nucleic acids that form complex structures (hairpins, ribozymes, etc.). We propose to continue with our single-molecule work on transcription by RNAP. Addition- ally, we plan to use a variant of the single-molecule assay for RNAP to address unsolved problems of co- transcriptional folding and gene regulation, particularly by riboswitches formed in nascent mRNAs, and to better understand the sequence elements that regulate transcriptional elongation and termination. A related assay will allow us to study the initiation of translation by ribosomes in eukaryotes, together with the sequence elements that modulate that process. Finally, we developed a successful single-molecule assay for transcriptional elongation by Pol II, the eukaryotic homolog of bacterial RNAP. We plan to capitalize on this opportunity by studying Pol II molecules purified from calf thymus and yeast, and to compare and contrast their biophysical properties with one another, as well as with those of prokaryotic RNAP. PUBLIC HEALTH RELEVANCE: Understanding the process of gene regulation is fundamental to any understanding of disease, because undesired changes in gene expression are responsible for the overwhelming majority of developmental and inherited disorders. Furthermore, many communicable diseases, especially those caused by human viruses, typically involve disruptions of gene regulation produced by the pathogen itself. The work described in this research proposal will supply new insights into the molecular basis of gene regulation by studying, at the single-molecule level, important gene- control molecules such as RNA polymerases, ribosomes, ribozymes, and riboswitches.
Funding Period: 1997-09-30 - 2014-11-30
more information: NIH RePORT

Top Publications

  1. pmc E. coli NusG inhibits backtracking and accelerates pause-free transcription by promoting forward translocation of RNA polymerase
    Kristina M Herbert
    Biophysics Program, Stanford University, Stanford, CA 94305, USA
    J Mol Biol 399:17-30. 2010
  2. pmc Direct observation of cotranscriptional folding in an adenine riboswitch
    Kirsten L Frieda
    Biophysics Program, Stanford University, Stanford, CA 94305, USA
    Science 338:397-400. 2012
  3. pmc Single-molecule studies of RNAPII elongation
    Jing Zhou
    Department of Applied Physics, Stanford University, Stanford, CA 94305, USA
    Biochim Biophys Acta 1829:29-38. 2013
  4. pmc Binding and translocation of termination factor rho studied at the single-molecule level
    Daniel J Koslover
    Biophysics Program, Stanford University, Stanford, CA 94305, USA
    J Mol Biol 423:664-76. 2012
  5. pmc Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II
    Matthew H Larson
    Biophysics Program, Stanford University, Stanford, CA 94305, USA
    Proc Natl Acad Sci U S A 109:6555-60. 2012
  6. pmc Folding energy landscape of the thiamine pyrophosphate riboswitch aptamer
    Peter C Anthony
    Biophysics Program, Department of Physics, Stanford University, Stanford, CA 94305, USA
    Proc Natl Acad Sci U S A 109:1485-9. 2012
  7. pmc Applied force provides insight into transcriptional pausing and its modulation by transcription factor NusA
    Jing Zhou
    Department of Applied Physics, Stanford University, Stanford, CA 94305, USA
    Mol Cell 44:635-46. 2011
  8. pmc Single-molecule studies of RNA polymerase: one singular sensation, every little step it takes
    Matthew H Larson
    Biophysics Program, Stanford University, Stanford, CA 94305, USA
    Mol Cell 41:249-62. 2011
  9. pmc Transcription factors TFIIF and TFIIS promote transcript elongation by RNA polymerase II by synergistic and independent mechanisms
    Volker Schweikhard
    Departments of Biology, Chemistry, Structural Biology, and Applied Physics, Stanford University, Stanford, CA 94305
    Proc Natl Acad Sci U S A 111:6642-7. 2014

Research Grants

  1. STRUCTURE AND FUNCTION OF NUCLEIC ACIDS
    Ignacio Tinoco; Fiscal Year: 2013

Detail Information

Publications11

  1. pmc E. coli NusG inhibits backtracking and accelerates pause-free transcription by promoting forward translocation of RNA polymerase
    Kristina M Herbert
    Biophysics Program, Stanford University, Stanford, CA 94305, USA
    J Mol Biol 399:17-30. 2010
    ....
  2. pmc Direct observation of cotranscriptional folding in an adenine riboswitch
    Kirsten L Frieda
    Biophysics Program, Stanford University, Stanford, CA 94305, USA
    Science 338:397-400. 2012
    ..Our results demonstrate that the outcome is kinetically controlled. These experiments furnish a means to observe conformational switching in real time and enable the precise mapping of events during cotranscriptional folding...
  3. pmc Single-molecule studies of RNAPII elongation
    Jing Zhou
    Department of Applied Physics, Stanford University, Stanford, CA 94305, USA
    Biochim Biophys Acta 1829:29-38. 2013
    ..This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation...
  4. pmc Binding and translocation of termination factor rho studied at the single-molecule level
    Daniel J Koslover
    Biophysics Program, Stanford University, Stanford, CA 94305, USA
    J Mol Biol 423:664-76. 2012
    ..These findings lead to a general model for Rho binding and translocation and establish a novel experimental approach that should facilitate additional single-molecule studies of RNA-binding proteins...
  5. pmc Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II
    Matthew H Larson
    Biophysics Program, Stanford University, Stanford, CA 94305, USA
    Proc Natl Acad Sci U S A 109:6555-60. 2012
    ....
  6. pmc Folding energy landscape of the thiamine pyrophosphate riboswitch aptamer
    Peter C Anthony
    Biophysics Program, Department of Physics, Stanford University, Stanford, CA 94305, USA
    Proc Natl Acad Sci U S A 109:1485-9. 2012
    ..We show that TPP binding proceeds in two steps, from a weakly to a strongly bound state. Our data imply a hierarchical folding sequence, and provide a framework for understanding molecular mechanism throughout the TPP riboswitch family...
  7. pmc Applied force provides insight into transcriptional pausing and its modulation by transcription factor NusA
    Jing Zhou
    Department of Applied Physics, Stanford University, Stanford, CA 94305, USA
    Mol Cell 44:635-46. 2011
    ..The effects of force and NusA on pause probabilities and lifetimes support a reaction scheme where nonbacktracked, elemental pauses branch off the elongation pathway from the pretranslocated state of RNAP...
  8. pmc Single-molecule studies of RNA polymerase: one singular sensation, every little step it takes
    Matthew H Larson
    Biophysics Program, Stanford University, Stanford, CA 94305, USA
    Mol Cell 41:249-62. 2011
    ..We focus on recent literature, highlighting examples where single-molecule methods have provided fresh insights into mechanism. We also present recent technological advances and outline future directions in the field...
  9. pmc Transcription factors TFIIF and TFIIS promote transcript elongation by RNA polymerase II by synergistic and independent mechanisms
    Volker Schweikhard
    Departments of Biology, Chemistry, Structural Biology, and Applied Physics, Stanford University, Stanford, CA 94305
    Proc Natl Acad Sci U S A 111:6642-7. 2014
    ..Overall, these experiments provide additional insights into how obstacles to transcription may be overcome by the concerted actions of multiple accessory factors. ..

Research Grants30

  1. STRUCTURE AND FUNCTION OF NUCLEIC ACIDS
    Ignacio Tinoco; Fiscal Year: 2013
    ..This information will lead to improved understanding of RNA structure, stability, and dynamics. It will help in understanding RNA function, and in controlling the role of RNA in human diseases. ..