Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Multiple folding pathways for the P4-P6 RNA domain.

S K Silverman1, M L Deras, S A Woodson

  • 1Department of Chemistry and Biochemistry and Howard Hughes Medical Institute, University of Colorado at Boulder, Boulder, Colorado 80309, USA. scott@scs.uiuc.edu

Biochemistry
|October 4, 2000
PubMed
Summary

Site-specific pyrene labeling monitors RNA tertiary structure folding. The P4-P6 domain of Tetrahymena group I intron RNA folds rapidly, but kinetics are sensitive to ionic strength and temperature, revealing complex folding pathways.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography.

Nature·2016
Same author

Dynamics of biological macromolecules: not a simple slaving by hydration water.

Biophysical journal·2010
Same author

Evolution of biological catalysis: ribozyme to RNP enzyme.

Cold Spring Harbor symposia on quantitative biology·2009
Same author

Translational careers.

Science (New York, N.Y.)·2009
Same author

Dynamics of tRNA at different levels of hydration.

Biophysical journal·2009
Same author

Probing RNA folding pathways by RNA fingerprinting.

Current protocols in nucleic acid chemistry·2008

Area of Science:

  • Biochemistry
  • Molecular Biology
  • RNA Structure and Dynamics

Background:

  • Site-specific labeling of RNA with pyrene enables monitoring of Mg(2+)-dependent tertiary structure formation.
  • Understanding RNA folding kinetics is crucial for elucidating biological function.

Purpose of the Study:

  • To investigate the folding kinetics of the Tetrahymena group I intron P4-P6 RNA domain using a novel pyrene labeling method.
  • To characterize the influence of temperature and ionic strength on RNA folding pathways.

Main Methods:

  • Stopped-flow fluorescence spectroscopy with millisecond time resolution.
  • Automated synthesis of pyrene-labeled oligoribonucleotides using a new phosphoramidite.
  • Synchrotron hydroxyl radical footprinting for validation.

Related Experiment Videos

Main Results:

  • The P4-P6 domain rapidly forms tertiary structure (t(1/2) ≈ 20–50 ms) at physiological conditions (35°C, 10 mM Mg(2+)).
  • Folding kinetics exhibit strong temperature dependence (high activation enthalpy) and are sensitive to ionic strength, with a slow folding phase observed at low ionic strength.
  • Hydroxyl radical footprinting confirms that fluorescence accurately reports on the same kinetic events.

Conclusions:

  • The P4-P6 domain folding is a rapid process, but exhibits complex kinetics influenced by solution conditions.
  • A model involving pre-existing tertiary interactions explains the observed fast and slow folding phases, dependent on ionic strength and temperature.
  • This study provides new insights into the dynamic nature of RNA tertiary structure formation.