Summary
The number and distribution of ribosomes on a transcript provide useful information in ascertaining the efficiency of translation. Herein we describe a direct method to determine the association of ribosomes with specific regions of an mRNA. The method, termed Ribosome Density Mapping (RDM), includes cleavage of ribosomes-associated mRNAs with RNase H and complementary oligodeoxynucleotide followed by separation of the cleavage products on a sucrose gradient. The gradient is then fractionated and the sedimentation position of each mRNA fragment is determined by northern analysis. Although developed for yeast mRNAs, RDM is likely to be applicable to various other systems.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sachs, M.S., Wang, Z., Gaba, A., Fang, P., Belk, J., Ganesan, R., Amrani, N. and Jacobson, A (2002) Toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mRNAs. Methods 26, 105–114.
Wolin, S.L. and Walter, P (1988) Ribosome pausing and stacking during translation of a eukaryotic mRNA. EMBO J 7, 3559–3569.
Wolin, S.L. and Walter, P (1989) Signal recognition particle mediates a transient elongation arrest of preprolactin in reticulocyte lysate. J Cell Biol 109, 2617–2622.
Mueller, P.P. and Hinnebusch, A.G (1986) Multiple upstream AUG codons mediate translational control of GCN4. Cell, 45, 201–207.
Abastado, J.P., Miller, P.F., Jackson, B.M. and Hinnebusch, A.G. (1991) Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis for GCN4 translational control. Mol Cell Biol 11, 486–496.
Werner, M., Feller, A., Messenguy, F. and Pierard, A (1987) The leader peptide of yeast gene CPA1 is essential for the translational repression of its expression. Cell 49, 805–813.
Sagliocco, F.A., Vega Laso, M.R., Zhu, D., Tuite, M.F., McCarthy, J.E. and Brown, A.J (1993) The influence of 5’-secondary structures upon ribosome binding to mRNA during translation in yeast. J Biol Chem 268, 26522–26530.
Yaman, I., Fernandez, J., Liu, H., Caprara, M., Komar, A.A., Koromilas, A.E., Zhou, L., Snider, M.D., Scheuner, D., Kaufman, R.J. et al (2003) The zipper model of translational control: a small upstream ORF is the switch that controls structural remodeling of an mRNA leader. Cell 113, 519–531.
Arava, Y., Boas, F.E., Brown, P.O. and Herschlag, D. (2005) Dissecting eukaryotic translation and its control by ribosome density mapping. Nucleic Acids Res 33, 2421–2432.
Arava, Y (2003) Isolation of polysomal RNA for microarray analysis. Methods Mol Biol 224, 79–87.
Kuhn, K.M., DeRisi, J.L., Brown, P.O. and Sarnow, P (2001) Global and specific translational regulation in the genomic response of Saccharomyces cerevisiae to a rapid transfer from a fermentable to a nonfermentable carbon source. Mol Cell Biol 21, 916–927.
Wang, Y., Liu, C.L., Storey, J.D., Tibshirani, R.J., Herschlag, D. and Brown, P.O (2002) Precision and functional specificity in mRNA decay. Proc Natl Acad Sci USA 99, 5860–5865.
Donis-Keller, H (1979) Site specific enzymatic cleavage of RNA. Nucleic Acids Res 7, 179–192.
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
Eldad, N., Arava, Y. (2008). A Ribosomal Density-Mapping Procedure to Explore Ribosome Positions Along Translating mRNAs. In: Wilusz, J. (eds) Post-Transcriptional Gene Regulation. Methods In Molecular Biology™, vol 419. Humana Press. https://doi.org/10.1007/978-1-59745-033-1_16
Download citation
DOI: https://doi.org/10.1007/978-1-59745-033-1_16
Publisher Name: Humana Press
Print ISBN: 978-1-58829-783-9
Online ISBN: 978-1-59745-033-1
eBook Packages: Springer Protocols