Kinetic intermediates trapped by native interactions in RNA folding

Science

Volumn 279, Issue 5358, 1998, Pages 1943-1946

  Treiber, Daniel K ^a   Rook, Martha S ^b   Zarrinkar, Patrick P ^b,c   Williamson, James R ^a  

^a SCRIPPS RESEARCH INSTITUTE   (United States)

^b MASSACHUSETTS INSTITUTE OF TECHNOLOGY   (United States)

^c DUKE UNIVERSITY MEDICAL CENTER   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

MAGNESIUM ION; RIBOZYME;

ARTICLE; GENE MUTATION; PRIORITY JOURNAL; RNA CONFORMATION; TETRAHYMENA;

ANIMALS; BINDING SITES; KINETICS; MAGNESIUM; MODELS, MOLECULAR; MUTATION; NUCLEIC ACID CONFORMATION; RNA, CATALYTIC; TETRAHYMENA;

TETRAHYMENA;

EID: 0032549735     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5358.1943     Document Type: Article

References (40)

1. P. P. Zarrinkar and J. R. Williamson, Science 265, 918 (1994).
2. P. P. Zarrinkar, J. Wang, J. R. Williamson, RNA 2, 564 (1996).
3. P. P. Zarrinkar and J. R. Williamson, Nature Struct. Biol. 3, 432 (1996).
4. B. Sclavi, M. Sullivan, M. R. Chance, M. Brenowitz, S. A. Woodson, Science 279, 1940 (1998); B. Sclavi, S. Woodson, M. Sullivan, M. R. Chance, M. Brenowitz, J. Mol. Biol. 266, 144 (1997).
5. B. Sclavi, M. Sullivan, M. R. Chance, M. Brenowitz, S. A. Woodson, Science 279, 1940 (1998); B. Sclavi, S. Woodson, M. Sullivan, M. R. Chance, M. Brenowitz, J. Mol. Biol. 266, 144 (1997).
6. A. R. Banerjee and D. H. Turner, Biochemistry 34, 6504 (1995).
7. W. D. Downs and T. R. Cech, RNA 2, 718 (1996).
8. K. M. Weeks and T. R. Cech, Science 271, 345 (1996).
9. C. D. Waldburger, T. Jonsson, R. T. Sauer, Proc. Natl. Acad. Sci. U.S.A. 93, 2629 (1996); M. Munson, K. S. Anderson, L. Regan, Folding Design 2, 77 (1997); D. M. Rothwarf and H. A. Scheraga, Biochemistry 35, 13797 (1996).
10. C. D. Waldburger, T. Jonsson, R. T. Sauer, Proc. Natl. Acad. Sci. U.S.A. 93, 2629 (1996); M. Munson, K. S. Anderson, L. Regan, Folding Design 2, 77 (1997); D. M. Rothwarf and H. A. Scheraga, Biochemistry 35, 13797 (1996).
11. C. D. Waldburger, T. Jonsson, R. T. Sauer, Proc. Natl. Acad. Sci. U.S.A. 93, 2629 (1996); M. Munson, K. S. Anderson, L. Regan, Folding Design 2, 77 (1997); D. M. Rothwarf and H. A. Scheraga, Biochemistry 35, 13797 (1996).
12. 2, 10 mM NaCl, 0.1 mM EDTA (disodium salt), and 1 mM dithiothreitol]. After 5 s at 37°C, RNAs with unfolded P3-P7 domains were cleaved selectively by incubation (30 s at 37°C) with oligonucleotide probes (60 μM) complementary to the 3′ strands of P3 (positions 268 to 281) and P7 (positions 303 to 316) and RNase H (U.S. Biochemical; final concentration, 0.1 U/μl) in 1 x folding buffer. Full-length RNAs were purified by denaturing polyacrylamide gel electrophoresis (PAGE). This treatment results in the cleavage of >85% of wild-type RNA. Both P3 and P7 probes were included to reduce the likelihood of selecting mutants that were simply mismatched with the probes. Step 4: Catalytically active RNAs that were not cleaved in step 3 were selectively copied by priming cDNA synthesis with an oligonucleotide complementary to the ligated substrate RNA. RNA from step 3 was incubated for 45 min at 37°C with 0.33 μM primer, 0.5 mM deoxynucleoside triphosphates, and murine leukemia virus reverse transcriptase (10 U/μ!) (U.S. Biochemical) in the supplied buffer. The resulting cDNA was amplified by the polymerase chain reaction with Taq polymerase (Perkin-Elmer).
13. 2, 10 mM NaCl, 0.1 mM EDTA (disodium salt), and 1 mM dithiothreitol]. After 5 s at 37°C, RNAs with unfolded P3-P7 domains were cleaved selectively by incubation (30 s at 37°C) with oligonucleotide probes (60 μM) complementary to the 3′ strands of P3 (positions 268 to 281) and P7 (positions 303 to 316) and RNase H (U.S. Biochemical; final concentration, 0.1 U/μl) in 1 x folding buffer. Full-length RNAs were purified by denaturing polyacrylamide gel electrophoresis (PAGE). This treatment results in the cleavage of >85% of wild-type RNA. Both P3 and P7 probes were included to reduce the likelihood of selecting mutants that were simply mismatched with the probes. Step 4: Catalytically active RNAs that were not cleaved in step 3 were selectively copied by priming cDNA synthesis with an oligonucleotide complementary to the ligated substrate RNA. RNA from step 3 was incubated for 45 min at 37°C with 0.33 μM primer, 0.5 mM deoxynucleoside triphosphates, and murine leukemia virus reverse transcriptase (10 U/μ!) (U.S. Biochemical) in the supplied buffer. The resulting cDNA was amplified by the polymerase chain reaction with Taq polymerase (Perkin-Elmer).
14. 9) and cloned individual molecules from the pool exhibited various degrees of biphasic folding kinetics. The fast phase generally accounted for 75 to 90% of the folding amplitude. Because the slow phase was minimal, single-exponential curve fitting provided a useful means for comparison; however, the rates for the fast and slow phases are likely to be underestimated and overestimated, respectively. Biphasic folding of cloned individual RNAs suggests that more than one pathway may lead to the folded state.
15. In general, the activation energy for P3-P7 formation is substantially lower for the fast folding mutants than for the wild type. Thus, at temperatures below 37°C, the rate of P3-P7 formation in some mutants is one to two orders of magnitude faster than that in the wild type (27).
16. V. Lehnert, L. Jaeger, F. Michel, E. Westhof, Chem. Biol. 3, 993 (1996).
17. J. H. Cate et al., Science 273, 1678 (1996); J. H. Cate et al., ibid., p. 1696; J. H. Cate, R. L. Hanna, J. A. Doudna, Nature Struct. Biol. 4, 553 (1997).
18. J. H. Cate et al., Science 273, 1678 (1996); J. H. Cate et al., ibid., p. 1696; J. H. Cate, R. L. Hanna, J. A. Doudna, Nature Struct. Biol. 4, 553 (1997).
19. J. H. Cate et al., Science 273, 1678 (1996); J. H. Cate et al., ibid., p. 1696; J. H. Cate, R. L. Hanna, J. A. Doudna, Nature Struct. Biol. 4, 553 (1997).
20. 2.
21. F. L. Murphy and T. R. Cech, Biochemistry 32, 5291 (1993); J. Mol. Biol. 236, 49 (1994).
22. F. L. Murphy and T. R. Cech, Biochemistry 32, 5291 (1993); J. Mol. Biol. 236, 49 (1994).
23. B. Laggerbauer, F. L. Murphy, T. R. Cech, EMBO J. 13, 2669 (1994).
24. 2 (15), but it has little effect on the stability of the full-length ribozyme (16). These results are consistent with our observation that the fast folding mutations have little effect on the stability of the folded structure (Table 1).
25. J. S. Weissman and P. S. Kim, Science 253, 1386 (1991).
26. J. Pan, D. Thirumalai, S. A. Woodson, J. Mol. Biol. 273, 7 (1997); T. Pan and T. R. Sosnick, Nature Struct. Biol. 4, 931 (1997).
27. J. Pan, D. Thirumalai, S. A. Woodson, J. Mol. Biol. 273, 7 (1997); T. Pan and T. R. Sosnick, Nature Struct. Biol. 4, 931 (1997).
28. Urea has little or no effect on the folding rates of the +G174, U167C, and A171G mutants, suggesting that these mutations also diminish the kinetic trap (27).
29. T. R. Sosnick, L. Mayne, R. Hiller, S. W. Englander, Nature Struct. Biol. 1, 149 (1994).
30. P. E. Cole, S. K. Yang, D. M. Crothers, Biochemistry 11, 4358 (1972).
31. S. E. Radford, C. M. Dobson, P. A. Evans, Nature 358, 302 (1992); A. Miranker, C. V. Robinson, S. E. Radford, R. T. Aplin, C. M. Dobson, Science 262 896 (1993).
32. S. E. Radford, C. M. Dobson, P. A. Evans, Nature 358, 302 (1992); A. Miranker, C. V. Robinson, S. E. Radford, R. T. Aplin, C. M. Dobson, Science 262 896 (1993).
33. G. Wildegger and T. Kiefhaber, J. Mol. Biol. 270, 294 (1997).
34. S. A. Woodson and T. R. Cech, Biochemistry 30, 2042 (1991).
35. D. E. Draper, Trends Biochem. Sci. 21, 145 (1996); D. Thirumalai and S. A. Woodson, Accounts Chem. Res. 29, 433 (1996); K. A. Dill and S. Chan, Nature Struct. Biol. 4, 10 (1997).
36. D. E. Draper, Trends Biochem. Sci. 21, 145 (1996); D. Thirumalai and S. A. Woodson, Accounts Chem. Res. 29, 433 (1996); K. A. Dill and S. Chan, Nature Struct. Biol. 4, 10 (1997).
37. D. E. Draper, Trends Biochem. Sci. 21, 145 (1996); D. Thirumalai and S. A. Woodson, Accounts Chem. Res. 29, 433 (1996); K. A. Dill and S. Chan, Nature Struct. Biol. 4, 10 (1997).
38. M. S. Rook, D. K. Treiber, J. R. Williamson, in preparation.
39. 2+, indicating that the mutations do not markedly destabilize the final, active conformation.
40. We thank P. Kim and B. Tidor for reviewing the manuscript, J. W. Orr for assistance with Fig. 2, and T.-R. Cech for providing A186U plasmid DNA. Supported by the Rita Allen Foundation, the Alfred P. Sloan Foundation, and the Camille and Henry Dreyfus Foundation (J.R.W.). D.K.T. was supported by an American Cancer Society postdoctoral fellowship and P.P.Z. by a Howard Hughes Medical Institute predoctoral fellowship.