Summary
Ribonuclease P (RNase P) from Escherichia coli is a transfer RNA (tRNA)-processing enzyme and consists of a catalytic RNA subunit (M1 RNA) and a protein component (C5 protein). M1GS, a gene-targeting ribozyme derived from M1 RNA, can cleave a target messenger RNA (mRNA) efficiently in vitro and inhibit its expression effectively in cultured cells. It has been shown that C5 protein can significantly increase the activities of M1 ribozyme and M1GS RNA in cleaving a natural tRNA substrate and a target mRNA, respectively. Understanding how C5 binds to M1GS RNA and affects the specific interactions between the ribozyme and its target mRNA substrates may facilitate the development of gene-targeting ribozymes that function effectively in vivo in the presence of cellular proteins. We describe the methods to determine the regions of a M1GS ribozyme that are potentially in close proximity to C5 protein. Specifically, methods are described in detail in using Fe(II)-ethylenediaminetetraacetic acid (EDTA) cleavage and nuclease footprint analyses to map the regions of the ribozyme in the absence and presence of C5 protein. These methods intend to provide experimental protocols for studying the regions of RNase P ribozyme that are in close contact with C5 protein.
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Acknowledgments
We thank Dr. Venkat Gopalan of Ohio State University for providing the purified C5 protein and Kihoon Kim for helpful discussions. P.T. was a recipient of the American Heart Association Predoctoral Fellowship (Western States Affiliate). F.L. was a Scholar of the Lymphoma and Leukemia Society of America and an Established Investigator of the American Heart Association. The research has been supported by the National Institutes of Health.
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Trang, P., Liu, F. (2008). Mapping the Regions of RNase P Catalytic RNA That Are Potentially in Close Contact With Its Protein Cofactor. In: Lin, RJ. (eds) RNA-Protein Interaction Protocols. Methods in Molecular Biology, vol 488. Humana Press. https://doi.org/10.1007/978-1-60327-475-3_19
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DOI: https://doi.org/10.1007/978-1-60327-475-3_19
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