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

Abstract

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.

Authors:
 [1];  [2];  [1];  [3];  [1]
+ Show Author Affiliations
  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
Publication Date:
Research Org.:
Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institute of General Medical Sciences; National Institute of Standards and Technology (NIST)
OSTI Identifier:
1170030
Grant/Contract Number:  
AC02-06CH11357
Resource Type:
Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 43; Journal Issue: 2; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
English

Citation Formats

Lee, Hui-Ting, Kilburn, D., Behrouzi, R., Briber, R. M., and Woodson, S. A. Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme. United States: N. p., 2014. Web. doi:10.1093/nar/gku1335.
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Lee, Hui-Ting, Kilburn, D., Behrouzi, R., Briber, R. M., & Woodson, S. A. Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme. United States. https://doi.org/10.1093/nar/gku1335
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Lee, Hui-Ting, Kilburn, D., Behrouzi, R., Briber, R. M., and Woodson, S. A. Fri . "Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme". United States. https://doi.org/10.1093/nar/gku1335. https://www.osti.gov/servlets/purl/1170030.
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@article{osti_1170030,
title = {Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme},
author = {Lee, Hui-Ting and Kilburn, D. and Behrouzi, R. and Briber, R. M. and Woodson, S. A.},
abstractNote = {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.},
doi = {10.1093/nar/gku1335},
journal = {Nucleic Acids Research},
number = 2,
volume = 43,
place = {United States},
year = {Fri Dec 05 00:00:00 EST 2014},
month = {Fri Dec 05 00:00:00 EST 2014}
}
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https://doi.org/10.1093/nar/gku1335
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Works referenced in this record:

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Molecular Crowding Favors Reactivity of a Human Ribozyme Under Physiological Ionic Conditions
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Structural Rearrangements Linked to Global Folding Pathways of the Azoarcus Group I Ribozyme
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Assembly of core helices and rapid tertiary folding of a small bacterial group I ribozyme
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Facilitation of RNA Enzyme Activity in the Molecular Crowding Media of Cosolutes
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The Molecular Interactions That Stabilize RNA Tertiary Structure: RNA Motifs, Patterns, and Networks
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Importance of Specific Nucleotides in the Folding of the Natural Form of the Hairpin Ribozyme †
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Crowders Perturb the Entropy of RNA Energy Landscapes to Favor Folding
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Roles of Long-Range Tertiary Interactions in Limiting Dynamics of the Tetrahymena Group I Ribozyme
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Dependence of RNA Tertiary Structural Stability on Mg 2+ Concentration: Interpretation of the Hill Equation and Coefficient
journal, March 2010

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  • Biochemistry, Vol. 49, Issue 9
  • DOI: 10.1021/bi902036j

Multistage Collapse of a Bacterial Ribozyme Observed by Time-Resolved Small-Angle X-ray Scattering
journal, July 2010

  • Roh, Joon Ho; Guo, Liang; Kilburn, J. Duncan
  • Journal of the American Chemical Society, Vol. 132, Issue 29
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Extended Structures in RNA Folding Intermediates Are Due to Nonnative Interactions Rather than Electrostatic Repulsion
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The Dynamic Landscapes of RNA Architecture
journal, February 2009

Cooperative Tertiary Interaction Network Guides RNA Folding
journal, April 2012

An optimal Mg2+ concentration for kinetic folding of the Tetrahymena ribozyme
journal, October 1999

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Molecular-crowding effects on single-molecule RNA folding/unfolding thermodynamics and kinetics
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Facilitation of Group I Splicing in Vivo: Misfolding of the Tetrahymena IVS and the Role of Ribosomal RNA Exons
journal, September 1999

  • Nikolcheva, Tania; Woodson, Sarah A.
  • Journal of Molecular Biology, Vol. 292, Issue 3
  • DOI: 10.1006/jmbi.1999.3083

Involvement of a GNRA tetraloop in long-range RNA tertiary interactions
journal, March 1994

The Dynamic Landscapes of RNA Architecture
journal, February 2009

Cooperative Tertiary Interaction Network Guides RNA Folding
journal, April 2012

Architecture and folding mechanism of the Azoarcus Group I Pre-tRNA
journal, May 2004

  • Rangan, Prashanth; Masquida, Benoı̂t; Westhof, Eric
  • Journal of Molecular Biology, Vol. 339, Issue 1
  • DOI: 10.1016/j.jmb.2004.03.059

Structural Rearrangements Linked to Global Folding Pathways of the Azoarcus Group I Ribozyme
journal, March 2009

  • Chauhan, Seema; Behrouzi, Reza; Rangan, Prashanth
  • Journal of Molecular Biology, Vol. 386, Issue 4
  • DOI: 10.1016/j.jmb.2008.12.075

Extended Structures in RNA Folding Intermediates Are Due to Nonnative Interactions Rather than Electrostatic Repulsion
journal, April 2010

  • Baird, Nathan J.; Gong, Haipeng; Zaheer, Syed S.
  • Journal of Molecular Biology, Vol. 397, Issue 5
  • DOI: 10.1016/j.jmb.2010.02.025

Structural Requirement for Mg2+ Binding in the Group I Intron Core
journal, May 2003

Cytoplasmic Degradation of Splice-Defective Pre-mRNAs and Intermediates
journal, December 2003

The Molecular Interactions That Stabilize RNA Tertiary Structure: RNA Motifs, Patterns, and Networks
journal, December 2011

  • Butcher, Samuel E.; Pyle, Anna Marie
  • Accounts of Chemical Research, Vol. 44, Issue 12
  • DOI: 10.1021/ar200098t

Importance of Specific Nucleotides in the Folding of the Natural Form of the Hairpin Ribozyme †
journal, February 2001

  • Wilson, Timothy J.; Zhao, Zheng-Yun; Maxwell, Kaera
  • Biochemistry, Vol. 40, Issue 7
  • DOI: 10.1021/bi002644p

Compaction of a Bacterial Group I Ribozyme Coincides with the Assembly of Core Helices †
journal, February 2004

  • Perez-Salas, Ursula A.; Rangan, Prashanth; Krueger, Susan
  • Biochemistry, Vol. 43, Issue 6
  • DOI: 10.1021/bi035642o

Molecular Crowding Favors Reactivity of a Human Ribozyme Under Physiological Ionic Conditions
journal, November 2013

  • Strulson, Christopher A.; Yennawar, Neela H.; Rambo, Robert P.
  • Biochemistry, Vol. 52, Issue 46
  • DOI: 10.1021/bi400816s

Dependence of RNA Tertiary Structural Stability on Mg 2+ Concentration: Interpretation of the Hill Equation and Coefficient
journal, March 2010

  • Leipply, Desirae; Draper, David E.
  • Biochemistry, Vol. 49, Issue 9
  • DOI: 10.1021/bi902036j

Assembly of an Exceptionally Stable RNA Tertiary Interface in a Group I Ribozyme †
journal, March 1999

  • Doherty, Elizabeth A.; Herschlag, Daniel; Doudna, Jennifer A.
  • Biochemistry, Vol. 38, Issue 10
  • DOI: 10.1021/bi982113p

Energetics and Cooperativity of Tertiary Hydrogen Bonds in RNA Structure †
journal, July 1999

  • Silverman, Scott K.; Cech, Thomas R.
  • Biochemistry, Vol. 38, Issue 27
  • DOI: 10.1021/bi9906118

Multistage Collapse of a Bacterial Ribozyme Observed by Time-Resolved Small-Angle X-ray Scattering
journal, July 2010

  • Roh, Joon Ho; Guo, Liang; Kilburn, J. Duncan
  • Journal of the American Chemical Society, Vol. 132, Issue 29
  • DOI: 10.1021/ja103867p

Crowders Perturb the Entropy of RNA Energy Landscapes to Favor Folding
journal, June 2013

  • Kilburn, Duncan; Roh, Joon Ho; Behrouzi, Reza
  • Journal of the American Chemical Society, Vol. 135, Issue 27
  • DOI: 10.1021/ja4030098

Roles of Long-Range Tertiary Interactions in Limiting Dynamics of the Tetrahymena Group I Ribozyme
journal, April 2014

  • Shi, Xuesong; Bisaria, Namita; Benz-Moy, Tara L.
  • Journal of the American Chemical Society, Vol. 136, Issue 18
  • DOI: 10.1021/ja413033d

Facilitation of RNA Enzyme Activity in the Molecular Crowding Media of Cosolutes
journal, November 2009

  • Nakano, Shu-ichi; Karimata, Hisae Tateishi; Kitagawa, Yuichi
  • Journal of the American Chemical Society, Vol. 131, Issue 46
  • DOI: 10.1021/ja9066628

Assembly of core helices and rapid tertiary folding of a small bacterial group I ribozyme
journal, February 2003

  • Rangan, P.; Masquida, B.; Westhof, E.
  • Proceedings of the National Academy of Sciences, Vol. 100, Issue 4
  • DOI: 10.1073/pnas.0337743100

Distinct contribution of electrostatics, initial conformational ensemble, and macromolecular stability in RNA folding
journal, April 2007

  • Laederach, A.; Shcherbakova, I.; Jonikas, M. A.
  • Proceedings of the National Academy of Sciences, Vol. 104, Issue 17
  • DOI: 10.1073/pnas.0608765104

Molecular-crowding effects on single-molecule RNA folding/unfolding thermodynamics and kinetics
journal, May 2014

  • Dupuis, N. F.; Holmstrom, E. D.; Nesbitt, D. J.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 23
  • DOI: 10.1073/pnas.1316039111

The Azoarcus Group I Intron Ribozyme Misfolds and Is Accelerated for Refolding by ATP-dependent RNA Chaperone Proteins
journal, August 2011

  • Sinan, Selma; Yuan, Xiaoyan; Russell, Rick
  • Journal of Biological Chemistry, Vol. 286, Issue 43
  • DOI: 10.1074/jbc.m111.287706

Increased Ribozyme Activity in Crowded Solutions
journal, December 2013

  • Desai, Ravi; Kilburn, Duncan; Lee, Hui-Ting
  • Journal of Biological Chemistry, Vol. 289, Issue 5
  • DOI: 10.1074/jbc.m113.527861

Ribonuclease P: The Evolution of an Ancient RNA Enzyme
journal, January 2006

  • Walker, Scott C.; Engelke, David R.
  • Critical Reviews in Biochemistry and Molecular Biology, Vol. 41, Issue 2
  • DOI: 10.1080/10409230600602634

Co-transcriptional commitment to alternative splice site selection
journal, December 1998

Analysis of the cooperative thermal unfolding of the td intron of bacteriophage T4
journal, June 1999

Structural Principles From Large RNAs
journal, June 2008

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