Journal of Molecular Biology
Volume 355, Issue 2, 13 January 2006, Pages 282-293
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Concordant Exploration of the Kinetics of RNA Folding from Global and Local Perspectives

https://doi.org/10.1016/j.jmb.2005.10.070Get rights and content

Time-resolved small-angle X-ray scattering (SAXS) with millisecond time-resolution reveals two discrete phases of global compaction upon Mg2+-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 (radical dotOH) 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.

Introduction

The addition of Mg2+ to the group I ribozyme from Tetrahymena thermophila induces folding to a biologically active conformation from an ensemble of states defined predominantly by secondary structure.1, 2, 3 The change in the global conformation of RNA during this process can be followed by small-angle X-ray scattering (SAXS), a technique that reports on the shape, size and conformational distribution of macromolecules.4 Time-resolved SAXS studies, conducted using a continuous-flow mixer, access global structural changes with millisecond time-resolution.5 SAXS analysis of the T. thermophila L-21 Sca1 ribozyme has shown that when Mg2+-mediated folding is initiated from a low ionic strength condition, the RNA ”relaxes” from an extended and rigid to a flexible and more compact ensemble of conformations; a second, slower transition is linked to the formation of five long-range tertiary contacts.5, 6 These long-range tertiary contacts are depicted in Figure 1. Prior studies monitoring local structure formation during Mg2+-mediated folding of the Tetrahymena ribozyme revealed a hierarchy of rates in the formation of the native tertiary structure of P4-P6 domain>periphery>catalytic core.7, 8

The present studies further explore the relationship between compaction and the formation of discrete tertiary interactions during RNA folding utilizing concordant measures of changes in global conformation and local tertiary structure. Hydroxyl radical (radical dotOH) footprinting is a well-established measure of changes in the solvent accessibility of the phosphodiester backbone of RNA with resolution as fine as a single nucleotide.9, 10 Synchrotron X-ray radical dotOH footprinting reports the time-evolution of discrete tertiary contacts also on the millisecond timescale.11, 12 Matched SAXS and radical dotOH footprinting analysis of wild-type ribozyme and judiciously selected mutants provide a definitive exploration of the kinetics of global compaction and tertiary contact formation during the Mg2+-mediated folding of RNA.

Since the P4-P6 domain is the first to fold upon the addition of Mg2+ from a very low salt initial condition,8 it is natural to question its role in the compaction kinetics. Experiments that probe the role of contacts within or exterior to the P4-P6 domain are thus a focus of our investigations. The data provide compelling evidence that: (i) the majority of tertiary contact formation occurs within a relaxed, screened, and moderately compact ensemble of molecules; (ii) the compaction due to tertiary contact formation depends on folding of the P4-P6 domain; and (iii) the reorganization of the misfolded catalytic core occurs within a highly constrained conformational space with either minor or no further compaction. The generality of these results and the importance of ambient ionic strength in cation-mediated RNA folding will be discussed.

Section snippets

Time-dependent global compaction

Global compaction of the ribozyme is assessed by calculating the molecule's time-dependent radius of gyration, Rg, from the angular dependence of X-rays scattered to the lowest angles. In the first set of studies, wild-type ribozyme and three mutants deficient in combinations of internal P4-P6 and peripheral tertiary contacts (Figure 1) were folded from an initial condition of 50 mM Mops (pH 7) by the addition of Mg2+ to a final concentration of 10 mM. The Rg of the wild-type ribozyme decreases

Discussion

This study presents an exceptional view of early events during the Mg2+-mediated folding of the Tetrahymena ribozyme from both global and local perspectives. The course of the SAXS progress curves following global compaction depend upon long-range tertiary contacts within the ribozyme as well as on ionic conditions of the folding reaction. One, two or three kinetic phases are present depending upon these variables. Time-resolved radical dotOH footprinting was used to follow the local changes in solvent

Conclusion

Concordant measures of global compaction and local changes in solvent accessibility provide a detailed portrait of the time-dependent relationship between compaction and tertiary contact formation during the Mg2+-mediated folding of a large RNA. The earliest monitored events, 1 ms after the initiation of folding, are dominated by the rapid formation of a compact and predominantly non-specifically collapsed ensemble of structures containing, at most, a small fraction of native and/or non-native

RNA preparation

Wild-type and four mutants of the L-21 ScaI ribozyme from T. thermophila25 were prepared by in vitro transcription as described.26 The double mutant ribozyme bears mutations of the A-rich bulge (AAUAAG (183–188) to UUUUUU) and the tetraloop receptor (UAAG (224–227) to AUA) destabilizing the major interhelix tertiary contacts within the P4-P6 domain.27 The triple mutant bears mutations of L2 (GGCAUGCACCU (39–49) to CUUCGGU), L9 (AAU (324–326) to C), and L9.1 (GAUGCAAC (346-353) to CUUCGG)

Acknowledgements

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

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