Abstract
Translation elongation is a highly choreographed process that involves substantial conformational changes of the ribosome to accommodate aminoacyl-tRNAs and traverse along the mRNA template. To capture distinct functional states of the ribosome, a high-resolution ribosome profiling-based approach has been developed. By deep-sequencing differently sized ribosome-protected mRNA fragments, this approach captures not only ribosome positions but also their functional states in vivo across the Saccharomyces cerevisiae transcriptome with codon resolution. This chapter presents a condensed and step-by-step protocol for preserving ribosomes in their functional states using a cocktail of antibiotics that traps distinct steps of elongating ribosomes and for constructing a cDNA library derived from the ribosome-protected mRNA fragments for deep sequencing.
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Acknowledgments
This protocol was initiated in the Green lab (Johns Hopkins University School of Medicine). The authors would like to thank Rachel Green for her support, and Nicholas R. Guydosh, Boris Zinshteyn, and Danny Nedialkova for helpful tips on developing this protocol. B.S.M. and M.S. are supported by Cancer Research Training Award. The work was support by the Intramural Research Program of the National Cancer Institute, National Institute of Health (C.C.-C.W.).
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Shafieinouri, M., Membreno, B.S., Wu, C.CC. (2022). High-Resolution Ribosome Profiling for Determining Ribosome Functional States During Translation Elongation. In: Matějů, D., Chao, J.A. (eds) The Integrated Stress Response. Methods in Molecular Biology, vol 2428. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1975-9_11
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DOI: https://doi.org/10.1007/978-1-0716-1975-9_11
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