Summary
Ultraviolet (UV)-dependent photochemical crosslinking is a powerful approach that can be used for the identification of RNA-protein interactions. Although 8-azidoATP (8-N3ATP) has been widely used to elucidate the ATP binding site of a variety of proteins, its inability to serve as an efficient substrate for bacteriophage RNA polymerases apparently restricted its actual potential as a photocrosslinking agent. In this chapter, in vitro transcription conditions that allow for template-dependent incorporation of 8-N3AMP into RNA are described. In addition, it is shown that a high-affinity MS2 coat protein binding sequence, in which adenosine residues were replaced by 8-azidoadenosine, crosslinks to the coat protein of the Escherichia coli phage MS2. This approach can be extended to identify almost any RNA binding protein.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Shapkina, T G., Dolan, M. A., Babin, P., and Wollenzien, P. (2000) Initiation factor 3-induced structural changes in the 30 S ribosomal subunit and in complexes containing tRNA(f)(Met) and mRNA. J. Mol. Biol. 299, 615–628.
Wu, S., and Green, M. R. (1997) Identification of a human protein that recognizes the 3′ splice site during the second step of pre-mRNA splicing. EMBO J. 16, 4421–4432.
Meisenheimer, K. M., Meisenheimer, P. L., Willis, M. C., and Koch, T. H. (1996) High yield photocrosslinking of a 5-iodocytidine (IC) substituted RNA to its associated protein. Nucleic Acids Res. 24, 981–982.
Willis, M. C., Hicke, B. J., Uhlenbeck, O. C., Cech, T. R., and Koch, T. H. (1993) Photocrosslinking of 5-iodouracil-substituted RNA and DNA to proteins. Science 262, 1255–1257.
Wang, Z., and Rana, T. M. (1998) RNA-protein interactions in the Tat-trans-acti-vation response element complex determined by site-specific photo-cross-linking. Biochemistry 37, 4235–4243.
Favre, A. (1990) In Morrison, H. (ed.), Bioorganic Photochemistry. J. Wiley & Sons, Inc., New York, Vol. 1.
Costas, C., Yuriev, E., Meyer, K. L., Guion, T. S., and Hanna, M. M. (2000) RNA-protein crosslinking to AMP residues at internal positions in RNA with a new photocrosslinking ATP analog. Nucleic Acids Res. 28, 1849–1858.
Sylvers, L. A., and Wower, J. (1993) Nucleic acid-incorporated azidonucleotides: probes for studying the interaction of RNA or DNA with proteins and other nucleic acids. Bioconjug. Chem. 4, 411–418.
Hanna, M. M. (1989) Photoaffinity cross-linking methods for studying RNA-protein interactions. Methods Enzymol. 180, 383–409.
Yu, Y. T. (1999) Construction of 4-thiouridine site-specifically substituted RNAs for cross-linking studies. Methods 18, 13–21.
Gaur, R. K., and Krupp, G. (1997) Chemical and enzymatic approaches to construct modified RNAs. Methods Mol. Biol. 74, 99–110.
Mundus, D., and Wollenzien, P. (2000) Structure determination by directed photo-cross-linking in large RNA molecules with site-specific psoralen. Methods Enzymol. 318, 104–118.
Milligan, J. F., Groebe, D. R., Witherell, G. W., and Uhlenbeck, O. C. (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15, 8783–8798.
Milligan, J. F., and Uhlenbeck, O. C. (1989) Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 180, 51–62.
Parang, K., Kohn, J. A., Saldanha, S. A., and Cole, P. A. (2002) Development of photo-crosslinking reagents for protein kinase-substrate interactions. FEBS Lett. 520, 156–160.
MacMillan, A. M., Query, C. C., Allerson, C. R., Chen, S., Verdine, G. L., and Sharp, P. A. (1994) Dynamic association of proteins with the pre-mRNA branch region. Genes Dev. 8, 3008–3020.
Potter, R. L., and Haley, B. E. (1983) Photoaffinity labeling of nucleotide binding sites with 8-azidopurine analogs: techniques and applications. Methods Enzymol. 91, 613–633.
Bowser, C. A., and Hanna, M. M. (1991) Sigma subunit of Escherichia coli RNA polymerase loses contacts with the 3′ end of the nascent RNA after synthesis of a tetranucleotide. J. Mol. Biol. 220, 227–239.
Gopalakrishna, S., Gusti, V., Nair, S., Sahar, S., and Gaur, R. K. (2004) Template-dependent incorporation of 8-N3AMP into RNA with bacteriophage T7 RNA polymerase. RNA 10, 1820–1830.
Kjems, J., Egebjerg, J., and Christiansen, J. (1998) Analysis of RNA-Protein Complexes In Vitro. Elsevier, Utrecht, The Netherlands, Vol. 26, pp. 13–53.
Krupp, G. (1989) Unusual promoter-independent transcription reactions with bacteriophage RNA polymerases. Nucleic Acids Res. 17, 3023–3036.
Pleiss, J. A., Derrick, M. L., and Uhlenbeck, O. C. (1998) T7 RNA polymerase produces 5′ end heterogeneity during in vitro transcription from certain templates. RNA 4, 1313–1317.
el-Deiry, W. S., So, A. G., and Downey, K. M. (1988) Mechanisms of error discrimination by Escherichia coli DNA polymerase I. Biochemistry 27, 546–553.
Conrad, F., Hanne, A., Gaur, R. K., and Krupp, G. (1995) Enzymatic synthesis of 2′-modified nucleic acids: identification of important phosphate and ribose moieties in RNase P substrates. Nucleic Acids Res. 23, 1845–1853.
Huang, Y., Beaudry, A., McSwiggen, J., and Sousa, R. (1997) Determinants of ribose specificity in RNA polymerization: effects of Mn2+ and deoxynucleoside monophosphate incorporation into transcripts. Biochemistry 36, 13718–13728.
Moore, M. J., and Sharp, P. A. (1992) Site-specific modification of pre-mRNA: the 2′-hydroxyl groups at the splice sites. Science 256, 992–997.
Query, C. C., Strobel, S. A., and Sharp, P. A. (1996) Three recognition events at the branch-site adenine. EMBO J. 15, 1392–1402.
Gozani, O., Patton, J. G., and Reed, R. (1994) A novel set of spliceosome-associ-ated proteins and the essential splicing factor PSF bind stably to pre-mRNA prior to catalytic step II of the splicing reaction. EMBO J. 13, 3356–3367.
Gaur, R. K., McLaughlin, L. W., and Green, M. R. (1997) Functional group substitutions of the branchpoint adenosine in a nuclear pre-mRNA and a group II intron. RNA 3, 861–869.
Acknowledgments
I would like to thank members of my laboratory. This work was supported in part by a grant from the Department of Defense (DAMD17-03-1-0625) and Beckman Research Institute excellence award.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science + Business Media, LLC
About this protocol
Cite this protocol
Gaur, R.K. (2008). T7 RNA Polymerase-Mediated Incorporation of 8-N3AMP Into RNA for Studying Protein-RNA Interactions. 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_11
Download citation
DOI: https://doi.org/10.1007/978-1-60327-475-3_11
Publisher Name: Humana Press
Print ISBN: 978-1-58829-419-7
Online ISBN: 978-1-60327-475-3
eBook Packages: Springer Protocols