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Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme

Journal Article · · Nucleic Acids Research
DOI:https://doi.org/10.1093/nar/gku1335· OSTI ID:1170030
 [1];  [2];  [1];  [3];  [1]
  1. Johns Hopkins Univ., Baltimore, MD (United States). Thomas C. Jenkins Dept. of Biophysics
  2. Johns Hopkins Univ., Baltimore, MD (United States). Thomas C. Jenkins Dept. of Biophysics; National Inst. of Standards and Technology, Gaithersburg, MD (United States). Center for Neutron Scattering Research
  3. Univ. of Maryland, College Park, MD (United States). Dept. of Materials Science and Engineering
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The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg2+ concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg2+ concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly.
Research Organization:
Advanced Photon Source (APS), Argonne National Laboratory (ANL), Argonne, IL (US)
Sponsoring Organization:
National Inst. of Standards and Technology; National Institute of General Medical Sciences; USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Grant/Contract Number:
AC02-06CH11357
OSTI ID:
1170030
Journal Information:
Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 2 Vol. 43; ISSN 0305-1048
Publisher:
Oxford University PressCopyright Statement
Country of Publication:
United States
Language:
English

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Cited By (8)

Optimal molecular crowding accelerates group II intron folding and maximizes catalysis journal November 2018
Structural Perspective on Revealing and Altering Molecular Functions of Genetic Variants Linked with Diseases journal December 2018
Crowders and Cosolvents-Major Contributors to the Cellular Milieu and Efficient Means to Counteract Environmental Stresses journal September 2017
Oligomerization of a Bimolecular Ribozyme Modestly Rescues its Structural Defects that Disturb Interdomain Assembly to Form the Catalytic Site journal August 2018
Lipid vesicles chaperone an encapsulated RNA aptamer journal June 2018
The roles of structural dynamics in the cellular functions of RNAs journal June 2019
Structural Perspective on Revealing and Altering Molecular Functions of Genetic Variants Linked with Diseases journal December 2018
Structural Perspective on Revealing and Altering Molecular Functions of Genetic Variants Linked with Diseases journal January 2019

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