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Volumn 288, Issue 5473, 2000, Pages 2048-2051

A single-molecule study of RNA catalysis and folding

Author keywords

[No Author keywords available]

Indexed keywords

RIBOZYME;

EID: 0034674420     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.288.5473.2048     Document Type: Article
Times cited : (665)

References (49)
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    • (1996) Nature , vol.380 , pp. 451
    • Vale, R.D.1
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    • M. D. Wang et al., Science 282, 902 (1998).
    • (1998) Science , vol.282 , pp. 902
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  • 12
    • 0003604405 scopus 로고
    • Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
    • R. F. Gesteland and J. F. Atkins, Eds., The RNA World (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1993).
    • (1993) The RNA World
    • Gesteland, R.F.1    Atkins, J.F.2
  • 23
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    • Supplementary material is available at www. sciencemag.org/feature/data/1049406.shl.
  • 24
    • 0343331801 scopus 로고    scopus 로고
    • note
    • To assess the specificity of ribozyme binding, streptavidin-coated surfaces were incubated with ribozyme bound to biotinylated and non-biotinylated tethers. The former gave 100-fold greater surface density of fluorescently labeled molecules than the latter, demonstrating that the biotinylated ribozyme binds to the surface predominately through the biotin-streptavidin interaction.
  • 25
    • 0343331802 scopus 로고    scopus 로고
    • note
    • In the total internal reflection microscope, fluorescent molecules were excited by an evanescent wave generated by total internal reflection of a 514-nm laser beam and detected by a CCD (charge-coupled device) camera (23). The microscope could detect several hundred individual molecules simultaneously with a time resolution of 100 ms. The cleavage kinetics and overall folding kinetics were measured using this apparatus. In the scanning confocal microscope, fluorescent molecules were excited by a focused 514-nm laser beam and detected by avalanche photodiodes. This apparatus can measure the fluorescence emission of one molecule with a time resolution of 1 ms. The docking kinetics were measured using this apparatus.
  • 27
    • 0342462594 scopus 로고    scopus 로고
    • note
    • 32P]-labeled S. After ≥1 min for binding of S, cleavage was initiated by addition of G. Reactions measuring the rate constant for S binding were carried out as previously described (26).
  • 33
    • 0343331800 scopus 로고    scopus 로고
    • note
    • o is typically between 3 and 7 nm, depending on the spectral overlap between the donor emission and acceptor absorption, quantum yield of the donor, and the orientations of the donor and acceptor. Thus, the FRET efficiency strongly depends on the distance between donor and acceptor as well as their orientations. Although there is a strong expectation of a large decrease in distance between the donor and acceptor upon Pl docking or overall folding, the conclusions here are independent of whether the FRET efficiency responds to changes in distance, orientation, or both.
  • 41
    • 0342896665 scopus 로고    scopus 로고
    • note
    • Previous studies of the rate constants for docking (35, 45), using different substrates under different conditions, yielded values within the same order of magnitude as reported here.
  • 48
    • 0342896664 scopus 로고    scopus 로고
    • note
    • -1, the same as the cleavage rate constant obtained from prefolded ribozyme.
  • 49
    • 0342462591 scopus 로고    scopus 로고
    • note
    • We are grateful to S. A. Scaringe and J. A. Orr for their advice on oligonucleotide preparation. This research was supported by an NSF grant (PHY-9970017) and an Air Force Office of Scientific Research grant (F49620-98-1-0219) to S.C. and an NIH grant (GM49423) to D.H. X.Z. was supported by the Chodorow Fellowship of the Stanford Applied Physics Department and an NIH postdoctoral fellowship. L.E.B. was supported in part by an NIH training grant. H.P.B. was supported in part by a Center on Polymer Interfaces and Macromolecular Assemblies grant. R.R. was supported by an NIH postdoctoral fellowship.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.