Abstract
Hydroxyl radical footprinting is a powerful technique often employed in characterization of the tertiary interactions between proteins and nucleic acids. Following the generation of a nucleic acid "ladder" either by chemical or enzymatic reactions, the radiolabeled products are traditionally separated by denaturing gel electrophoresis and further quantified by phosphorimaging techniques. Here we report the use of ion pair reverse phase liquid chromatography to analyze the products of an RNA footprinting reaction using fluorescently labeled RNA molecules. This technique offers several advantages over existing procedures, including rapid analysis, automation, and direct quantification of the cleavage products without the need to employ radiolabeling. To illustrate the resolving power of this technique, we have analyzed the products of base hydrolysis, generated from a fluorescently labeled RNA molecule and have subsequently used this method to define the solvent accessibility of the substrate strand as it docks with the hairpin ribozyme.
Full Text
The Full Text of this article is available as a PDF (244.0 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Celander D. W., Cech T. R. Visualizing the higher order folding of a catalytic RNA molecule. Science. 1991 Jan 25;251(4992):401–407. doi: 10.1126/science.1989074. [DOI] [PubMed] [Google Scholar]
- Earnshaw D. J., Masquida B., Müller S., Sigurdsson S. T., Eckstein F., Westhof E., Gait M. J. Inter-domain cross-linking and molecular modelling of the hairpin ribozyme. J Mol Biol. 1997 Nov 28;274(2):197–212. doi: 10.1006/jmbi.1997.1405. [DOI] [PubMed] [Google Scholar]
- Fedor M. J. Structure and function of the hairpin ribozyme. J Mol Biol. 2000 Mar 24;297(2):269–291. doi: 10.1006/jmbi.2000.3560. [DOI] [PubMed] [Google Scholar]
- Hampel A., Cowan J. A. A unique mechanism for RNA catalysis: the role of metal cofactors in hairpin ribozyme cleavage. Chem Biol. 1997 Jul;4(7):513–517. doi: 10.1016/s1074-5521(97)90323-9. [DOI] [PubMed] [Google Scholar]
- Hampel K. J., Walter N. G., Burke J. M. The solvent-protected core of the hairpin ribozyme-substrate complex. Biochemistry. 1998 Oct 20;37(42):14672–14682. doi: 10.1021/bi981083n. [DOI] [PubMed] [Google Scholar]
- Latham J. A., Cech T. R. Defining the inside and outside of a catalytic RNA molecule. Science. 1989 Jul 21;245(4915):276–282. doi: 10.1126/science.2501870. [DOI] [PubMed] [Google Scholar]
- Nesbitt S., Hegg L. A., Fedor M. J. An unusual pH-independent and metal-ion-independent mechanism for hairpin ribozyme catalysis. Chem Biol. 1997 Aug;4(8):619–630. doi: 10.1016/s1074-5521(97)90247-7. [DOI] [PubMed] [Google Scholar]
- Rupert P. B., Ferré-D'Amaré A. R. Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis. Nature. 2001 Apr 12;410(6830):780–786. doi: 10.1038/35071009. [DOI] [PubMed] [Google Scholar]
- Weeks K. M., Cech T. R. Protein facilitation of group I intron splicing by assembly of the catalytic core and the 5' splice site domain. Cell. 1995 Jul 28;82(2):221–230. doi: 10.1016/0092-8674(95)90309-7. [DOI] [PubMed] [Google Scholar]
- Wu J. C., Kozarich J. W., Stubbe J. The mechanism of free base formation from DNA by bleomycin. A proposal based on site specific tritium release from Poly(dA.dU). J Biol Chem. 1983 Apr 25;258(8):4694–4697. [PubMed] [Google Scholar]
- Young K. J., Gill F., Grasby J. A. Metal ions play a passive role in the hairpin ribozyme catalysed reaction. Nucleic Acids Res. 1997 Oct 1;25(19):3760–3766. doi: 10.1093/nar/25.19.3760. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Young K. J., Vyle J. S., Pickering T. J., Cohen M. A., Holmes S. C., Merkel O., Grasby J. A. The role of essential pyrimidines in the hairpin ribozyme-catalysed reaction. J Mol Biol. 1999 May 21;288(5):853–866. doi: 10.1006/jmbi.1999.2748. [DOI] [PubMed] [Google Scholar]