Identifying kinetic barriers to mechanical unfolding of the T. thermophila ribozyme

Science

Volumn 299, Issue 5614, 2003, Pages 1892-1895

  Onoa, Bibiana ^a,c   Dumont, Sophie ^a   Liphardt, Jan ^a,b   Smith, Steven B ^a   Tinoco Jr , Ignacio ^a,b   Bustamante, Carlos ^a,b  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b LAWRENCE BERKELEY NATIONAL LABORATORY   (United States)

^c DUPONT COMPANY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

COMPLEXATION; MOLECULAR BIOLOGY; RNA;

KINETIC BARRIERS;

ENZYME KINETICS;

MAGNESIUM; RIBOZYME;

MICROBIOLOGY; RNA;

ARTICLE; ENERGY; GENETIC STABILITY; MOLECULAR DYNAMICS; MOLECULAR INTERACTION; MOLECULAR SIZE; NONHUMAN; PRIORITY JOURNAL; PROTEIN DOMAIN; PROTEIN FOLDING; RNA TRANSLATION; RNA TRANSPORT; STOCHASTIC MODEL; TETRAHYMENA THERMOPHILA; TRANSPORT KINETICS; VIRUS REPLICATION;

ANIMALS; CATALYTIC DOMAIN; KINETICS; MAGNESIUM; MUTATION; NUCLEIC ACID CONFORMATION; OLIGONUCLEOTIDES, ANTISENSE; RNA, CATALYTIC; TETRAHYMENA THERMOPHILA; THERMODYNAMICS;

PROTOZOA; TETRAHYMENA THERMOPHILA;

EID: 0242417558     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.1081338     Document Type: Article

References (33)

1. J. Liphardt, B. Onoa, S. B. Smith, I. Tinoco Jr., C. Bustamante, Science 292, 733 (2001).
2. S. B. Smith, Y. Cui, C. Bustamante, Methods Enzymol. 361, 134 (2003).
3. Materials and methods are available as supporting material on Science Online.
4. X. Zhuang et al., Science 288, 2048 (2000).
5. R. Russell et al., Proc. Natl. Acad. Sci. U.S.A. 99, 155 (2002).
6. The L-21 derivative of this molecule lacks its native substrates (exons) but catalyzes the cleavage of substrate RNAs provided in trans.
7. A. J. Zaug, C. A. Grosshans, T. R. Cech, Biochemistry 27, 8924 (1988).
8. D. K. Treiber, J. R. Williamson, Curr. Opin. Struct. Biol. 11, 309 (2001).
9. B. L. Golden, A. R. Gooding, E. R. Podell, T. R. Cech, Science 282, 259 (1998).
10. V. Lehnert, L. Jaeger, F. Michel, E. Westhof, Chem. Biol. 3, 993 (1996).
11. D. Thirumalai, N. Lee, S. A. Woodson, D. K. Klimov, Ann. Rev. Phys. Chem. 52, 751 (2001).
12. U. Bockelmann, B. Essavez-Roulet, F. Heslot, Phys. Rev. E 58, 286 (1998).
13. U. Bockelmann, P. Thomen, B. Essevaz-Roulet, V. Viasnoff, F. Heslot, Biophys. J. 82, 1537 (2002).
14. U. Gerland, R. Bundschuh, T. Hwa, Biophys. J. 81, 1324 (2001).
15. _, Biophys. J., in press; preprint available at http://arxiv.org/abs/cond-mat/0208202.
16. I. Tinoco Jr., C. Bustamante, J. Mol. Biol. 293, 271 (1999).
17. C. Bustamante, J. F. Marko, E. D. Siggia, S. B. Smith, Science 265, 1599 (1994).
18. J. H. Cate et al., Science 273, 1678 (1996).
19. K. Juneau, E, Podell, D. J. Harrington, T. R. Cech, Structure 9, 221 (2001).
20. B. Sclavi, M. Sullivan, M. R. Chance, M. Brenowitz, S. A. Woodson, Science 279, 1940 (1998).
21. P. P. Zarrinkar, J. R. Williamson, Science 265, 918 (1994).
22. C. Y. Ralston, Q. He, M. Brenowitz, M. R. Chance, Nature Struct. Biol. 7, 371 (2000).
23. The reasons for choosing a waiting time of 30 s at 2 pN are as follows: In vitro and under similar ionic and temperature conditions, P4-P6 folds at a rate of 4.7 s-1. Here, we let P4-P6 refold for 30 s (two orders of magnitude longer than stated above) to favor complete refolding of the molecule. We compared P4-P6 refolding at 0, 2, 4, and 6 pN and observed no substantial differences in unfolding curves. We chose 2 pN because it allowed complete refolding but minimized nonspecific interactions and the attachment of additional molecules between the two beads.
24. Mechanical unfolding of P4-P6 after 0 s (732 curves) and 30 s (323 curves) of refolding both displayed three barriers. The barriers were within 5 nt of each other, the frequencies of arrest at the barriers were within 7% of each other, and the rupture forces were within 2 pN of each other.
25. S. K. Silverman, M. L. Deras, S. A. Woodson, S. A. Scaringe, T. R. Cech, Biochemistry 39, 12465 (2000).
26. Independent of the context of the barriers, their rupture forces and position are constant to within 2 pN and 5 nt, respectively. For example, P5abc ruptures at 19 pN in the context of both P4-P6 (Fig. 2C) and L-21 (Fig. 4B) and releases 70 nt in P4-P6 and 73 in L-21.
27. The lengths of rips b and c change between L-21Δp2 (Fig. 3) and L-21 (Fig. 4A). Rip b is longer than c in L-21ΔP2, and rip c is longer than b in L-21. When P2 is added to L-21Δp2 to yield L-21, the P13 interaction now takes place between P9.1a and P2.1, causing P9.1a to rupture at the same time as P9 instead of at the same time as P9.1.
28. Similar lengths for motifs P9, P9.1, P9.1a, P.2, and P2.1, and their interactions (P13, P14, and T4; Fig. 1A), make precise localization of barriers b, c, and d difficult, However, from our analysis we propose that barrier b corresponds to P9.1; barrier c, to P9-P9.1a; and barrier d, to P2-P2.1. Site-directed mutagenesis and/or varying the lengths of these motifs could help verify the proposed barrier localizations.
29. Without the 30-s refolding period, the frequencies of arrest at barriers b, c, and d are all below 0.0S and thus lower than the frequency of arrest at barrier e (catalytic core). In contrast, with the 30-s refolding period (Fig. 4B), frequencies of arrest at those barriers are comparable to frequency of arrest at barrier e.
30. Barriers b to h involve secondary and tertiary contacts, with the exception of P9.1, which does not make tertiary contacts but has a high GC content. Data not shown here revealed that a high GC content is associated with high rupture forces and a lack of detectable bistability in current experimental conditions (31).
31. J. Liphardt, unpublished observations.
32. R. T. Batey, R. P. Rambo, J. A. Doudna, Angew. Chem. Int. Ed. 38, 2326 (1999).
33. This research was supported in part by NIH grants GM-10840 and GM-32543, Department of Energy grants DE-FG03-86ER60406 and DE-AC03-76DF00098, and NSF grants MBC-9118482 and DBI-9732140. B.O. and J.L. were supported by the Program in Mathematics and Molecular Biology through a Burroughs Wellcome Fund Fellowship, and S.D. is supported by the Natural Sciences and Engineering Research Council of Canada and Fonds pour la Formation de Chercheurs et l'Aide à la Recherche du Québec. We thank J. McCoy for work with L-21 mutants; P. Gorostiza for helpful suggestions; and S. Woodson for encouragement, helpful discussions, and multiple constructs.