Abstract
Endoribonuclease footprinting is an important technique for probing RNA–protein interactions with single nucleotide resolution. The susceptibility of RNA residues to enzymatic digestion gives information about the RNA secondary structure, the location of protein binding sites, and the effects of protein binding on the RNA structure. Here we present a detailed protocol for using RNase T2, which cleaves single stranded RNA with a preference for A nucleotides, to footprint the protein Hfq on the rpoS mRNA leader. This protocol covers how to form the RNP complex, determine the correct dose of enzyme, footprint the protein, and analyze the cleavage pattern using primer extension.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Gottesman S, Storz G (2011) Bacterial small RNA regulators: versatile roles and rapidly evolving variations. Cold Spring Harb Perspect Biol. 2011 Dec 1;3(12). pii: a003798. doi: 10.1101/cshperspect.a003798
Valentin-Hansen P, Eriksen M, Udesen C (2004) The bacterial Sm-like protein Hfq: a key player in RNA transactions. Mol Microbiol 51:1525–1533
Zhang A, Wassarman KM, Ortega J, Steven AC, Storz G (2002) The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs. Mol Cell 9:11–22
Salim NN, Feig AL (2010) An upstream Hfq binding site in the fhlA mRNA leader region facilitates the OxyS-fhlA interaction. PLoS One 5:e13028
Moller T, Franch T, Hojrup P, Keene DR, Bachinger HP, Brennan RG, Valentin-Hansen P (2002) Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction. Mol Cell 9:23–30
Soper TJ, Woodson SA (2008) The rpoS mRNA leader recruits Hfq to facilitate annealing with DsrA sRNA. RNA 14:1907–1917
Soper TJ, Doxzen K, Woodson SA (2011) Major role for mRNA binding and restructuring in sRNA recruitment by Hfq. RNA 17:1544–1550
Ruskin B, Green MR (1985) Specific and stable intron-factor interactions are established early during in vitro pre-mRNA splicing. Cell 43:131–142
Brescia CC, Mikulecky PJ, Feig AL, Sledjeski DD (2003) Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure. RNA 9:33–43
Lease RA, Woodson SA (2004) Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA. J Mol Biol 344:1211–1223
Geissmann TA, Touati D (2004) Hfq, a new chaperoning role: binding to messenger RNA determines access for small RNA regulator. EMBO J 23:396–405
Parker KA, Steitz JA (1989) Determination of RNA-protein and RNA-ribonucleoprotein interactions by nuclease probing. Methods Enzymol 180:454–468
Ehresmann C, Baudin F, Mougel M, Romby P, Ebel JP, Ehresmann B (1987) Probing the structure of RNAs in solution. Nucleic Acids Res 15:9109–9128
Lease RA, Cusick ME, Belfort M (1998) Riboregulation in Escherichia coli: DsrA RNA acts by RNA:RNA interactions at multiple loci. Proc Natl Acad Sci U S A 95:12456–12461
Majdalani N, Cunning C, Sledjeski D, Elliott T, Gottesman S (1998) DsrA RNA regulates translation of RpoS message by an anti-antisense mechanism, independent of its action as an antisilencer of transcription. Proc Natl Acad Sci U S A 95:12462–12467
Mitra S, Shcherbakova IV, Altman RB, Brenowitz M, Laederach A (2008) High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis. Nucleic Acids Res 36:e63
Vasa SM, Guex N, Wilkinson KA, Weeks KM, Giddings MC (2008) ShapeFinder: a software system for high-throughput quantitative analysis of nucleic acid reactivity information resolved by capillary electrophoresis. RNA 14:1979–1990
Brenowitz M, Senear DF, Shea MA, Ackers GK (1986) Quantitative DNase footprint titration: a method for studying protein-DNA interactions. Methods Enzymol 130:132–181
Abramoff MD, Magalhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophotonics Int 11:36–42
Das R, Laederach A, Pearlman SM, Herschlag D, Altman RB (2005) SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments. RNA 11:344–354
Soper T, Mandin P, Majdalani N, Gottesman S, Woodson SA (2010) Positive regulation by small RNAs and the role of Hfq. Proc Natl Acad Sci U S A 107:9602–9607
Hopkins JF, Panja S, Woodson SA (2011) Rapid binding and release of Hfq from ternary complexes during RNA annealing. Nucleic Acids Res 39:5193–5202
Zaug AJ, Grosshans CA, Cech TR (1988) Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes. Biochemistry 27:8924–8931
Acknowledgement
We would like to acknowledge Richard Lease, Subrata Panja, and Sarah Soper for providing reagents, technical advice, and useful discussions.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Peng, Y., Soper, T.J., Woodson, S.A. (2012). RNase Footprinting of Protein Binding Sites on an mRNA Target of Small RNAs. In: Keiler, K. (eds) Bacterial Regulatory RNA. Methods in Molecular Biology, vol 905. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-949-5_13
Download citation
DOI: https://doi.org/10.1007/978-1-61779-949-5_13
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-948-8
Online ISBN: 978-1-61779-949-5
eBook Packages: Springer Protocols