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

Lee, Hui-Ting; Kilburn, D.; Behrouzi, R.; Briber, R. M.; Woodson, S. A.

doi:10.1093/nar/gku1335

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

Journal Article · Fri Dec 05 00:00:00 EST 2014 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gku1335· OSTI ID:1170030

Lee, Hui-Ting ^[1]; Kilburn, D. ^[2]; Behrouzi, R. ^[1]; Briber, R. M. ^[3]; Woodson, S. A. ^[1]

Johns Hopkins Univ., Baltimore, MD (United States). Thomas C. Jenkins Dept. of Biophysics
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
Univ. of Maryland, College Park, MD (United States). Dept. of Materials Science and Engineering

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.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institute of General Medical Sciences; National Institute of Standards and Technology (NIST)

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 1170030

Journal Information:: Nucleic Acids Research, Vol. 43, Issue 2; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: English

Citation Metrics:

Cited by: 22 works

Citation information provided by
Web of Science

References (47)

Assembly of an Exceptionally Stable RNA Tertiary Interface in a Group I Ribozyme ^† Doherty, Elizabeth A.; Herschlag, Daniel; Doudna, Jennifer A. Biochemistry, Vol. 38, Issue 10 https://doi.org/10.1021/bi982113p	journal	March 1999
Molecular Crowding Favors Reactivity of a Human Ribozyme Under Physiological Ionic Conditions Strulson, Christopher A.; Yennawar, Neela H.; Rambo, Robert P. Biochemistry, Vol. 52, Issue 46 https://doi.org/10.1021/bi400816s	journal	November 2013
Determination of the regularization parameter in indirect-transform methods using perceptual criteria Svergun, D. I. Journal of Applied Crystallography, Vol. 25, Issue 4, p. 495-503 https://doi.org/10.1107/S0021889892001663	journal	August 1992
Compaction of a Bacterial Group I Ribozyme Coincides with the Assembly of Core Helices ^† Perez-Salas, Ursula A.; Rangan, Prashanth; Krueger, Susan Biochemistry, Vol. 43, Issue 6 https://doi.org/10.1021/bi035642o	journal	February 2004
Intracellular folding of the Tetrahymena group I intron depends on exon sequence and promoter choice Koduvayur, S. P. RNA, Vol. 10, Issue 10 https://doi.org/10.1261/rna.7880404	journal	October 2004
The kinetic mechanism of the hairpin ribozyme in vivo : influence of RNA helix stability on intracellular cleavage kinetics 1 1Edited by D. E. Draper Donahue, Christine P.; Yadava, Ramesh S.; Nesbitt, Steven M. Journal of Molecular Biology, Vol. 295, Issue 3 https://doi.org/10.1006/jmbi.1999.3380	journal	January 2000
Fast folding mutants of the Tetrahymena group I ribozyme reveal a rugged folding energy landscape 1 1Edited by D. Draper Rook, Martha S.; Treiber, Daniel K.; Williamson, James R. Journal of Molecular Biology, Vol. 281, Issue 4 https://doi.org/10.1006/jmbi.1998.1960	journal	August 1998
Metal Ion Dependence of Cooperative Collapse Transitions in RNA Moghaddam, Sarvin; Caliskan, Gokhan; Chauhan, Seema Journal of Molecular Biology, Vol. 393, Issue 3 https://doi.org/10.1016/j.jmb.2009.08.044	journal	October 2009
Structural Principles From Large RNAs Holbrook, Stephen R. Annual Review of Biophysics, Vol. 37, Issue 1 https://doi.org/10.1146/annurev.biophys.36.040306.132755	journal	June 2008
Structural Rearrangements Linked to Global Folding Pathways of the Azoarcus Group I Ribozyme Chauhan, Seema; Behrouzi, Reza; Rangan, Prashanth Journal of Molecular Biology, Vol. 386, Issue 4 https://doi.org/10.1016/j.jmb.2008.12.075	journal	March 2009
Involvement of a GNRA tetraloop in long-range RNA tertiary interactions Jaeger, Luc; Michel, François; Westhof, Eric Journal of Molecular Biology, Vol. 236, Issue 5 https://doi.org/10.1016/0022-2836(94)90055-8	journal	March 1994
Assembly of core helices and rapid tertiary folding of a small bacterial group I ribozyme Rangan, P.; Masquida, B.; Westhof, E. Proceedings of the National Academy of Sciences, Vol. 100, Issue 4 https://doi.org/10.1073/pnas.0337743100	journal	February 2003
Distinct contribution of electrostatics, initial conformational ensemble, and macromolecular stability in RNA folding Laederach, A.; Shcherbakova, I.; Jonikas, M. A. Proceedings of the National Academy of Sciences, Vol. 104, Issue 17 https://doi.org/10.1073/pnas.0608765104	journal	April 2007
RNA Tertiary Interactions Mediate Native Collapse of a Bacterial Group I Ribozyme Chauhan, Seema; Caliskan, Gokhan; Briber, Robert M. Journal of Molecular Biology, Vol. 353, Issue 5 https://doi.org/10.1016/j.jmb.2005.09.015	journal	November 2005
Macromolecular crowding: obvious but underappreciated Ellis, R. John Trends in Biochemical Sciences, Vol. 26, Issue 10 https://doi.org/10.1016/S0968-0004(01)01938-7	journal	October 2001
Energetics and Cooperativity of Tertiary Hydrogen Bonds in RNA Structure ^† Silverman, Scott K.; Cech, Thomas R. Biochemistry, Vol. 38, Issue 27 https://doi.org/10.1021/bi9906118	journal	July 1999
Mg ²⁺ -Dependent Compaction and Folding of Yeast tRNA ^Phe and the Catalytic Domain of the B. subtilis RNase P RNA Determined by Small-Angle X-ray Scattering ^† Fang, Xingwang; Littrell, Kenneth; Yang, Xiao-jing Biochemistry, Vol. 39, Issue 36 https://doi.org/10.1021/bi000724n	journal	September 2000
Catalytic Activity as a Probe of Native RNA Folding Wan, Yaqi; Mitchell III, David; Russell, Rick Methods in Enzymology https://doi.org/10.1016/S0076-6879(09)68010-1	book	November 2009
Molecular Crowding Stabilizes Folded RNA Structure by the Excluded Volume Effect Kilburn, Duncan; Roh, Joon Ho; Guo, Liang Journal of the American Chemical Society, Vol. 132, Issue 25 https://doi.org/10.1021/ja101500g	journal	June 2010
Architecture and folding mechanism of the Azoarcus Group I Pre-tRNA Rangan, Prashanth; Masquida, Benoı̂t; Westhof, Eric Journal of Molecular Biology, Vol. 339, Issue 1 https://doi.org/10.1016/j.jmb.2004.03.059	journal	May 2004
Facilitation of RNA Enzyme Activity in the Molecular Crowding Media of Cosolutes Nakano, Shu-ichi; Karimata, Hisae Tateishi; Kitagawa, Yuichi Journal of the American Chemical Society, Vol. 131, Issue 46 https://doi.org/10.1021/ja9066628	journal	November 2009
Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site Herschlag, Daniel; Cech, Thomas R. Biochemistry, Vol. 29, Issue 44 https://doi.org/10.1021/bi00496a003	journal	November 1990
Analysis of the cooperative thermal unfolding of the td intron of bacteriophage T4 Brion, P. Nucleic Acids Research, Vol. 27, Issue 12 https://doi.org/10.1093/nar/27.12.2494	journal	June 1999
Multiple native states reveal persistent ruggedness of an RNA folding landscape Solomatin, Sergey V.; Greenfeld, Max; Chu, Steven Nature, Vol. 463, Issue 7281 https://doi.org/10.1038/nature08717	journal	February 2010
The Molecular Interactions That Stabilize RNA Tertiary Structure: RNA Motifs, Patterns, and Networks Butcher, Samuel E.; Pyle, Anna Marie Accounts of Chemical Research, Vol. 44, Issue 12 https://doi.org/10.1021/ar200098t	journal	December 2011
Long-range tertiary interactions in single hammerhead ribozymes bias motional sampling toward catalytically active conformations McDowell, S. E.; Jun, J. M.; Walter, N. G. RNA, Vol. 16, Issue 12 https://doi.org/10.1261/rna.1829110	journal	October 2010
Importance of Specific Nucleotides in the Folding of the Natural Form of the Hairpin Ribozyme ^† Wilson, Timothy J.; Zhao, Zheng-Yun; Maxwell, Kaera Biochemistry, Vol. 40, Issue 7 https://doi.org/10.1021/bi002644p	journal	February 2001
Crowders Perturb the Entropy of RNA Energy Landscapes to Favor Folding Kilburn, Duncan; Roh, Joon Ho; Behrouzi, Reza Journal of the American Chemical Society, Vol. 135, Issue 27 https://doi.org/10.1021/ja4030098	journal	June 2013
Ribonuclease P: The Evolution of an Ancient RNA Enzyme Walker, Scott C.; Engelke, David R. Critical Reviews in Biochemistry and Molecular Biology, Vol. 41, Issue 2 https://doi.org/10.1080/10409230600602634	journal	January 2006
Structural basis for altering the stability of homologous RNAs from a mesophilic and a thermophilic bacterium Baird, N. J. RNA, Vol. 12, Issue 4 https://doi.org/10.1261/rna.2186506	journal	February 2006
Maximizing RNA folding rates: A balancing act Thirumalai, D.; Woodson, Sarah A. RNA, Vol. 6, Issue 6 https://doi.org/10.1017/S1355838200000522	journal	June 2000
Facilitation of Group I Splicing in Vivo: Misfolding of the Tetrahymena IVS and the Role of Ribosomal RNA Exons Nikolcheva, Tania; Woodson, Sarah A. Journal of Molecular Biology, Vol. 292, Issue 3 https://doi.org/10.1006/jmbi.1999.3083	journal	September 1999
Co-transcriptional commitment to alternative splice site selection Roberts, G. Nucleic Acids Research, Vol. 26, Issue 24 https://doi.org/10.1093/nar/26.24.5568	journal	December 1998
Roles of Long-Range Tertiary Interactions in Limiting Dynamics of the Tetrahymena Group I Ribozyme Shi, Xuesong; Bisaria, Namita; Benz-Moy, Tara L. Journal of the American Chemical Society, Vol. 136, Issue 18 https://doi.org/10.1021/ja413033d	journal	April 2014
Dependence of RNA Tertiary Structural Stability on Mg ²⁺ Concentration: Interpretation of the Hill Equation and Coefficient Leipply, Desirae; Draper, David E. Biochemistry, Vol. 49, Issue 9 https://doi.org/10.1021/bi902036j	journal	March 2010
Multistage Collapse of a Bacterial Ribozyme Observed by Time-Resolved Small-Angle X-ray Scattering Roh, Joon Ho; Guo, Liang; Kilburn, J. Duncan Journal of the American Chemical Society, Vol. 132, Issue 29 https://doi.org/10.1021/ja103867p	journal	July 2010
Extended Structures in RNA Folding Intermediates Are Due to Nonnative Interactions Rather than Electrostatic Repulsion Baird, Nathan J.; Gong, Haipeng; Zaheer, Syed S. Journal of Molecular Biology, Vol. 397, Issue 5 https://doi.org/10.1016/j.jmb.2010.02.025	journal	April 2010
The Dynamic Landscapes of RNA Architecture Cruz, José Almeida; Westhof, Eric Cell, Vol. 136, Issue 4 https://doi.org/10.1016/j.cell.2009.02.003	journal	February 2009
Cooperative Tertiary Interaction Network Guides RNA Folding Behrouzi, Reza; Roh, Joon Ho; Kilburn, Duncan Cell, Vol. 149, Issue 2 https://doi.org/10.1016/j.cell.2012.01.057	journal	April 2012
An optimal Mg2+ concentration for kinetic folding of the Tetrahymena ribozyme Rook, M. S.; Treiber, D. K.; Williamson, J. R. Proceedings of the National Academy of Sciences, Vol. 96, Issue 22 https://doi.org/10.1073/pnas.96.22.12471	journal	October 1999
Molecular-crowding effects on single-molecule RNA folding/unfolding thermodynamics and kinetics Dupuis, N. F.; Holmstrom, E. D.; Nesbitt, D. J. Proceedings of the National Academy of Sciences, Vol. 111, Issue 23 https://doi.org/10.1073/pnas.1316039111	journal	May 2014
Tertiary Interactions Determine the Accuracy of RNA Folding Chauhan, Seema; Woodson, Sarah A. Journal of the American Chemical Society, Vol. 130, Issue 4 https://doi.org/10.1021/ja076166i	journal	January 2008
Frequent use of the same tertiary motif by self-folding RNAs. Costa, M.; Michel, F. The EMBO Journal, Vol. 14, Issue 6 https://doi.org/10.1002/j.1460-2075.1995.tb07111.x	journal	March 1995
Structural Requirement for Mg2+ Binding in the Group I Intron Core Rangan, Prashanth; Woodson, Sarah A. Journal of Molecular Biology, Vol. 329, Issue 2 https://doi.org/10.1016/s0022-2836(03)00430-3	journal	May 2003
Cytoplasmic Degradation of Splice-Defective Pre-mRNAs and Intermediates Hilleren, Patricia J.; Parker, Roy Molecular Cell, Vol. 12, Issue 6 https://doi.org/10.1016/s1097-2765(03)00488-x	journal	December 2003
The Azoarcus Group I Intron Ribozyme Misfolds and Is Accelerated for Refolding by ATP-dependent RNA Chaperone Proteins Sinan, Selma; Yuan, Xiaoyan; Russell, Rick Journal of Biological Chemistry, Vol. 286, Issue 43 https://doi.org/10.1074/jbc.m111.287706	journal	August 2011
Increased Ribozyme Activity in Crowded Solutions Desai, Ravi; Kilburn, Duncan; Lee, Hui-Ting Journal of Biological Chemistry, Vol. 289, Issue 5 https://doi.org/10.1074/jbc.m113.527861	journal	December 2013

Cited By (8)

Crowders and Cosolvents-Major Contributors to the Cellular Milieu and Efficient Means to Counteract Environmental Stresses Gao, Mimi; Held, Christoph; Patra, Satyajit ChemPhysChem, Vol. 18, Issue 21 https://doi.org/10.1002/cphc.201700762	journal	September 2017
Oligomerization of a Bimolecular Ribozyme Modestly Rescues its Structural Defects that Disturb Interdomain Assembly to Form the Catalytic Site Rahman, Md. Motiar; Matsumura, Shigeyoshi; Ikawa, Yoshiya Journal of Molecular Evolution, Vol. 86, Issue 7 https://doi.org/10.1007/s00239-018-9862-8	journal	August 2018
Lipid vesicles chaperone an encapsulated RNA aptamer Saha, Ranajay; Verbanic, Samuel; Chen, Irene A. Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-018-04783-8	journal	June 2018
The roles of structural dynamics in the cellular functions of RNAs Ganser, Laura R.; Kelly, Megan L.; Herschlag, Daniel Nature Reviews Molecular Cell Biology, Vol. 20, Issue 8 https://doi.org/10.1038/s41580-019-0136-0	journal	June 2019
Structural Perspective on Revealing and Altering Molecular Functions of Genetic Variants Linked with Diseases Peng, Yunhui; Alexov, Emil; Basu, Sankar International Journal of Molecular Sciences, Vol. 20, Issue 3 https://doi.org/10.3390/ijms20030548	journal	January 2019
Structural Perspective on Revealing and Altering Molecular Functions of Genetic Variants Linked with Diseases Peng, Yunhui; Alexov, Emil; Basu, Sankar International Journal of Molecular Sciences, Vol. 20 https://doi.org/10.20944/preprints201812.0273.v2	journal	December 2018
Optimal molecular crowding accelerates group II intron folding and maximizes catalysis Paudel, Bishnu P.; Fiorini, Erica; Börner, Richard Proceedings of the National Academy of Sciences, Vol. 115, Issue 47 https://doi.org/10.1073/pnas.1806685115	journal	November 2018
Structural Perspective on Revealing and Altering Molecular Functions of Genetic Variants Linked with Diseases Peng, Yunhui; Alexov, Emil; Basu, Sankar International Journal of Molecular Sciences, Vol. 20 https://doi.org/10.20944/preprints201812.0273.v3	journal	December 2018

Similar Records

Compaction of a Bacterial Group I Ribozyme Coincides with the Assembly of Core Helices

Journal Article · Thu Jan 01 00:00:00 EST 2004 · Biochemistry · OSTI ID:1170030

Perez-Salas, U; Rangan, P; Krueger, S; +3 more

Effects of molecular crowding on a bimolecular group I ribozyme and its derivative that self-assembles to form ribozyme oligomers

Journal Article · Sat Dec 15 00:00:00 EST 2018 · Biochemical and Biophysical Research Communications · OSTI ID:1170030

Rahman, Md Motiar; Matsumura, Shigeyoshi; Ikawa, Yoshiya

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

Journal Article · Tue Aug 03 00:00:00 EDT 2010 · J. Am. Chem. Soc. · OSTI ID:1170030

Roh, Joon Ho; Guo, Liang; Kilburn, J Duncan; +3 more

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

Citation Formats

References (47)

Cited By (8)

Similar Records

Related Subjects