Concordant exploration of the kinetics of RNA folding from global and local perspectives

Lisa W. Kwok; Inna Shcherbakova; Jessica S. Lamb; Hye Yoon Park; Kurt Andresen; Heather Smith; Michael Brenowitz; Lois Pollack

doi:10.1016/j.jmb.2005.10.070

Concordant exploration of the kinetics of RNA folding from global and local perspectives

Lisa W. Kwok
, Inna Shcherbakova
, Jessica S. Lamb
, Hye Yoon Park
, Kurt Andresen
, Heather Smith
, Michael Brenowitz
, Lois Pollack

Research output: Contribution to journal › Article › peer-review

56 Scopus citations

Abstract

Time-resolved small-angle X-ray scattering (SAXS) with millisecond time-resolution reveals two discrete phases of global compaction upon Mg 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_g) decreases from 75 Å to 55 Å. A further decrease in R_g to 45 Å 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 Å compaction transition. A better understanding of the role of specific tertiary contacts in compaction was obtained by concordant time-resolved hydroxyl radical (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_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.

Original language	English (US)
Pages (from-to)	282-293
Number of pages	12
Journal	Journal of Molecular Biology
Volume	355
Issue number	2
DOIs	https://doi.org/10.1016/j.jmb.2005.10.070
State	Published - Jan 13 2006
Externally published	Yes

Bibliographical note

Funding Information:
This work was supported by grant P01-GM066275 from the National Institute of General Medical Sciences. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. W-31-109-Eng-38. We acknowledge additional support from NASA under contract number NAG8-1778 (to L.P.), and from the National Science Foundation through grant MCB-0347220 (to L.P.) and the Cornell Nanobiotechnology Center. This work is also based upon research conducted at the Cornell High Energy Synchrotron Source (CHESS), which is supported by the National Science Foundation and the National Institutes of Health/National Institute of General Medical Sciences under award DMR-0225180. The X-ray footprinting studies were conducted at beamline X-28C of the National Synchrotron Light Source supported by grant P41-EB0001979 from the National Institute of Biomedical Imaging and Bioengineering. Fabrication of the SAXS mixing devices was conducted in the Cornell Nanoscale Science and Technology Facility that is supported by the NSF, Cornell University and industrial affiliates. We thank Sayan Gupta for his support of beamline X-28C operations, Simon Mochrie for the generous loan of a stopped-flow mixer, Alec Sandy and Suresh Narayanan for their assistance at beamline 8-IDI at the APS, and Arthur Woll and Ernie Fontes for assistance at the CHESS G-1 beamline. We thank Rhiju Das and Dan Herschlag for helpful discussions.

Keywords

Compaction
Electrostatic relaxation
RNA folding
Tertiary structure formation
Time-resolved small-angle X-ray scattering

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10.1016/j.jmb.2005.10.070

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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 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 (Rg) decreases from 75 {\AA} to 55 {\AA}. A further decrease in Rg to 45 {\AA} 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 {\AA} compaction transition. A better understanding of the role of specific tertiary contacts in compaction was obtained by concordant time-resolved hydroxyl radical (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 Rg 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.",

keywords = "Compaction, Electrostatic relaxation, RNA folding, Tertiary structure formation, Time-resolved small-angle X-ray scattering",

author = "Kwok, \{Lisa W.\} and Inna Shcherbakova and Lamb, \{Jessica S.\} and Park, \{Hye Yoon\} and Kurt Andresen and Heather Smith and Michael Brenowitz and Lois Pollack",

note = "Funding Information: This work was supported by grant P01-GM066275 from the National Institute of General Medical Sciences. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. W-31-109-Eng-38. We acknowledge additional support from NASA under contract number NAG8-1778 (to L.P.), and from the National Science Foundation through grant MCB-0347220 (to L.P.) and the Cornell Nanobiotechnology Center. This work is also based upon research conducted at the Cornell High Energy Synchrotron Source (CHESS), which is supported by the National Science Foundation and the National Institutes of Health/National Institute of General Medical Sciences under award DMR-0225180. The X-ray footprinting studies were conducted at beamline X-28C of the National Synchrotron Light Source supported by grant P41-EB0001979 from the National Institute of Biomedical Imaging and Bioengineering. Fabrication of the SAXS mixing devices was conducted in the Cornell Nanoscale Science and Technology Facility that is supported by the NSF, Cornell University and industrial affiliates. We thank Sayan Gupta for his support of beamline X-28C operations, Simon Mochrie for the generous loan of a stopped-flow mixer, Alec Sandy and Suresh Narayanan for their assistance at beamline 8-IDI at the APS, and Arthur Woll and Ernie Fontes for assistance at the CHESS G-1 beamline. We thank Rhiju Das and Dan Herschlag for helpful discussions.",

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doi = "10.1016/j.jmb.2005.10.070",

language = "English (US)",

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T1 - Concordant exploration of the kinetics of RNA folding from global and local perspectives

AU - Kwok, Lisa W.

AU - Shcherbakova, Inna

AU - Lamb, Jessica S.

AU - Park, Hye Yoon

AU - Andresen, Kurt

AU - Smith, Heather

AU - Brenowitz, Michael

AU - Pollack, Lois

N1 - Funding Information: This work was supported by grant P01-GM066275 from the National Institute of General Medical Sciences. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. W-31-109-Eng-38. We acknowledge additional support from NASA under contract number NAG8-1778 (to L.P.), and from the National Science Foundation through grant MCB-0347220 (to L.P.) and the Cornell Nanobiotechnology Center. This work is also based upon research conducted at the Cornell High Energy Synchrotron Source (CHESS), which is supported by the National Science Foundation and the National Institutes of Health/National Institute of General Medical Sciences under award DMR-0225180. The X-ray footprinting studies were conducted at beamline X-28C of the National Synchrotron Light Source supported by grant P41-EB0001979 from the National Institute of Biomedical Imaging and Bioengineering. Fabrication of the SAXS mixing devices was conducted in the Cornell Nanoscale Science and Technology Facility that is supported by the NSF, Cornell University and industrial affiliates. We thank Sayan Gupta for his support of beamline X-28C operations, Simon Mochrie for the generous loan of a stopped-flow mixer, Alec Sandy and Suresh Narayanan for their assistance at beamline 8-IDI at the APS, and Arthur Woll and Ernie Fontes for assistance at the CHESS G-1 beamline. We thank Rhiju Das and Dan Herschlag for helpful discussions.

PY - 2006/1/13

Y1 - 2006/1/13

N2 - Time-resolved small-angle X-ray scattering (SAXS) with millisecond time-resolution reveals two discrete phases of global compaction upon Mg 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 (Rg) decreases from 75 Å to 55 Å. A further decrease in Rg to 45 Å 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 Å compaction transition. A better understanding of the role of specific tertiary contacts in compaction was obtained by concordant time-resolved hydroxyl radical (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 Rg 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.

AB - Time-resolved small-angle X-ray scattering (SAXS) with millisecond time-resolution reveals two discrete phases of global compaction upon Mg 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 (Rg) decreases from 75 Å to 55 Å. A further decrease in Rg to 45 Å 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 Å compaction transition. A better understanding of the role of specific tertiary contacts in compaction was obtained by concordant time-resolved hydroxyl radical (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 Rg 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.

KW - Compaction

KW - Electrostatic relaxation

KW - RNA folding

KW - Tertiary structure formation

KW - Time-resolved small-angle X-ray scattering

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UR - https://www.scopus.com/pages/publications/28844509318#tab=citedBy

U2 - 10.1016/j.jmb.2005.10.070

DO - 10.1016/j.jmb.2005.10.070

M3 - Article

C2 - 16303138

AN - SCOPUS:28844509318

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VL - 355

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EP - 293

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

IS - 2

ER -

Concordant exploration of the kinetics of RNA folding from global and local perspectives

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Bibliographical note

Keywords

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