skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Concordant Exploration of the Kinetics of RNA Folding from Global and Local Perspectives

Abstract

Time-resolved small-angle X-ray scattering (SAXS) with millisecond time-resolution reveals two discrete phases of global compaction upon Mg{sup 2+}-mediated folding of the Tetrahymena thermophila ribozyme. Electrostatic relaxation of the RNA occurs rapidly and dominates the first phase of compaction during which the observed radius of gyration (R{sub g}) decreases from 75 Angstroms to 55 Angstroms. A further decrease in R{sub g} to 45 Angstroms occurs in a well-defined second phase. An analysis of mutant ribozymes shows that the latter phase depends upon the formation of long-range tertiary contacts within the P4-P6 domain of the ribozyme; disruption of the three remaining long-range contacts linking the peripheral helices has no effect on the 55-45 Angstroms compaction transition. A better understanding of the role of specific tertiary contacts in compaction was obtained by concordant time-resolved hydroxyl radical ({center_dot}OH) analyses that report local changes in the solvent accessibility of the RNA backbone. Comparison of the global and local measures of folding shows that formation of a subset of native tertiary contacts (i.e. those defining the ribozyme core) can occur within a highly compact ensemble whose R{sub g} is close to that of the fully folded ribozyme. Analyses of additional ribozyme mutants and reaction conditions establishmore » the generality of the rapid formation of a partially collapsed state with little to no detectable tertiary structure. These studies directly link global RNA compaction with formation of tertiary structure as the molecule acquires its biologically active structure, and underscore the strong dependence on salt of both local and global measures of folding kinetics.« less

Authors:
; ; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
914343
Report Number(s):
BNL-78911-2007-JA
Journal ID: ISSN 0022-2836; JMOBAK; TRN: US200809%%185
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Mol. Biol.; Journal Volume: 355
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ELECTROSTATICS; EXPLORATION; HYDROXYL RADICALS; KINETICS; MUTANTS; RELAXATION; RNA; SCATTERING; SOLVENTS; TETRAHYMENA; national synchrotron light source

Citation Formats

Kwok,L., Scherbakova, I., Lamb, J., Park, H., Andresen, K., Smith, H., Brenowitz, M., and Pollack, L.. Concordant Exploration of the Kinetics of RNA Folding from Global and Local Perspectives. United States: N. p., 2006. Web. doi:10.1016/j.jmb.2005.10.070.
Kwok,L., Scherbakova, I., Lamb, J., Park, H., Andresen, K., Smith, H., Brenowitz, M., & Pollack, L.. Concordant Exploration of the Kinetics of RNA Folding from Global and Local Perspectives. United States. doi:10.1016/j.jmb.2005.10.070.
Kwok,L., Scherbakova, I., Lamb, J., Park, H., Andresen, K., Smith, H., Brenowitz, M., and Pollack, L.. Sun . "Concordant Exploration of the Kinetics of RNA Folding from Global and Local Perspectives". United States. doi:10.1016/j.jmb.2005.10.070.
@article{osti_914343,
title = {Concordant Exploration of the Kinetics of RNA Folding from Global and Local Perspectives},
author = {Kwok,L. and Scherbakova, I. and Lamb, J. and Park, H. and Andresen, K. and Smith, H. and Brenowitz, M. and Pollack, L.},
abstractNote = {Time-resolved small-angle X-ray scattering (SAXS) with millisecond time-resolution reveals two discrete phases of global compaction upon Mg{sup 2+}-mediated folding of the Tetrahymena thermophila ribozyme. Electrostatic relaxation of the RNA occurs rapidly and dominates the first phase of compaction during which the observed radius of gyration (R{sub g}) decreases from 75 Angstroms to 55 Angstroms. A further decrease in R{sub g} to 45 Angstroms occurs in a well-defined second phase. An analysis of mutant ribozymes shows that the latter phase depends upon the formation of long-range tertiary contacts within the P4-P6 domain of the ribozyme; disruption of the three remaining long-range contacts linking the peripheral helices has no effect on the 55-45 Angstroms compaction transition. A better understanding of the role of specific tertiary contacts in compaction was obtained by concordant time-resolved hydroxyl radical ({center_dot}OH) analyses that report local changes in the solvent accessibility of the RNA backbone. Comparison of the global and local measures of folding shows that formation of a subset of native tertiary contacts (i.e. those defining the ribozyme core) can occur within a highly compact ensemble whose R{sub g} is close to that of the fully folded ribozyme. Analyses of additional ribozyme mutants and reaction conditions establish the generality of the rapid formation of a partially collapsed state with little to no detectable tertiary structure. These studies directly link global RNA compaction with formation of tertiary structure as the molecule acquires its biologically active structure, and underscore the strong dependence on salt of both local and global measures of folding kinetics.},
doi = {10.1016/j.jmb.2005.10.070},
journal = {J. Mol. Biol.},
number = ,
volume = 355,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}
  • At the heart of the RNA folding problem is the number, structures, and relationships among the intermediates that populate the folding pathways of most large RNA molecules. Unique insight into the structural dynamics of these intermediates can be gleaned from the time-dependent changes in local probes of macromolecular conformation (e.g. reports on individual nucleotide solvent accessibility offered by hydroxyl radical ({center_dot}OH) footprinting). Local measures distributed around a macromolecule individually illuminate the ensemble of separate changes that constitute a folding reaction. Folding pathway reconstruction from a multitude of these individual measures is daunting due to the combinatorial explosion of possible kineticmore » models as the number of independent local measures increases. Fortunately, clustering of time progress curves sufficiently reduces the dimensionality of the data so as to make reconstruction computationally tractable. The most likely folding topology and intermediates can then be identified by exhaustively enumerating all possible kinetic models on a super-computer grid. The folding pathways and measures of the relative flux through them were determined for Mg{sup 2+} and Na{sup +}-mediated folding of the Tetrahymena thermophila group I intron using this combined experimental and computational approach. The flux during Mg{sup 2+}-mediated folding is divided among numerous parallel pathways. In contrast, the flux during the Na{sup +}-mediated reaction is predominantly restricted through three pathways, one of which is without detectable passage through intermediates. Under both conditions, the folding reaction is highly parallel with no single pathway accounting for more than 50% of the molecular flux. This suggests that RNA folding is non-sequential under a variety of different experimental conditions even at the earliest stages of folding. This study provides a template for the systematic analysis of the time-evolution of RNA structure from ensembles of local measures that will illuminate the chemical and physical characteristics of each step in the process. The applicability of this analysis approach to other macromolecules is discussed.« less
  • No abstract prepared.
  • The model age or ‘date of purification’ of a nuclear material is an important nuclear forensic signature. In this study, chemical separation and MC-ICP-MS measurement techniques were developed for 226 Ra and 227Ac: grand-daughter nuclides in the 238U and 235U decay chains respectively. The 230Th- 234U, 226Ra- 238U, 231Pa- 235U, and 227Ac- 235U radiochronometers were used to calculate model ages for CRM-U100 standard reference material and two highly-enriched pieces of uranium metal from the International Technical Working Group Round Robin 3 Exercise. In conclusion, the results demonstrate the accuracy of the 226Ra- 238U and 227Ac- 235U chronometers and provide informationmore » about nuclide migration during uranium processing.« less
  • No abstract prepared.