Abstract
Photochemical crosslinking is a powerful technique for characterization of RNA-protein interactions in ribonucleoprotein complexes. Intermolecular crosslinks can be generated without chemical modification of either the RNA or the protein by irradiation of native complexes with short wavelength ultraviolet (UV) light (254 nm). Covalent crosslinks form when the nucleotides or amino acids in the RNA and protein are photochemically converted to reactive species. This method has the advantage of allowing the study of interactions in which neither macromolecule has been modified, which can, in some cases, alter the way that the protein and RNA make contact. However, this approach produces very low crosslinking yields and often leads to nicking of the RNA and degradation of some proteins. This photodamage to the RNA and protein makes subsequent mapping of the position(s) of the crosslink(s) at the nucleotide and amino acid level difficult. Crosslinking by this method is also relatively nonspecific, making localization of the protein contacts to a single nucleotide or region in the RNA fairly difficult.
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References
Hanna, M. M. (1989) Photoaffinity crosslinking methods for studying RNA-Protein interactions. Methods Enzymol. RNA Proc. 180, 383–409.
Hanna, M. M., Dissinger, S., Williams, B. D., and Colston, J. E. (1989) Synthesis and characterization of 5-((4-azidophenacyl)thio)-uridine-5′-triphosphate: a cleavable photocrosslinking nucleotide analog. Biochemistry 28, 5814–5820.
Dissinger, S. and Hanna, M. M. (1990) Active site labeling of E. coli transcription complexes with 5-((4-azidophenacyl)thio)-uridine-5′-triphosphate. J. Biol. Chem. 265, 7662–7668.
He, B. and Hanna, M. M. (1995) Preparation of probe-modified RNA with 5-mercapto-UTP for analysis of protein-RNA Interactions. Nucleic Acids Res. 73, 1231–1238.
Hanna, M. M., Zhang, Y., Reidling, J. C., Thomas, M. J., and Jou, J. (1993) Synthesis and characterization of a new photocrosslinking CTP analog and its use in photoaffinity labeling E. coli and T7 RNA polymerases. Nucleic Acids Res. 21, 2073–2079.
Liu, K. and Hanna, M. M. (1995) NusA interferes with interactions between the nascent RNA and the C-terminal domain of the alpha subunit in E. coli transcription complexes. Proc. Natl. Acad. Sci. USA 92, 5012–5016.
Liu, K. and Hanna, M. M. (1995) NusA contacts nascent RNA in E. coli transcription complexes. J. Mol. Biol. 247, 547–558.
Zhang, Y. and Hanna, M. (1994) NusA changes the conformation of E. coli RNA polymerase at the binding site for the 3′ end of the nascent RNA. J. Bacteriol. 176, 1787–1789.
Schmidt, M. and Hanna, M. M. (1986) Nonenzymatic radiolabeling of protein by 32P-containing nucleotides. FEBS Lett. 194, 305–308.
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© 1999 Humana Press Inc.
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Hanna, M.M., Bentsen, L., Lucido, M., Sapre, A. (1999). RNA-Protein Crosslinking with Photoreactive Nucleotide Analogs. In: Haynes, S.R. (eds) RNA-Protein Interaction Protocols. Methods in Molecular Biology™, vol 118. Humana Press. https://doi.org/10.1385/1-59259-676-2:21
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DOI: https://doi.org/10.1385/1-59259-676-2:21
Publisher Name: Humana Press
Print ISBN: 978-0-89603-568-3
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