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High-Resolution Ribosome Profiling for Determining Ribosome Functional States During Translation Elongation

  1. Author:
    Shafieinouri,Mohammad
    Santiago Membreno,Britnie
    Wu,Colin

  2. Author Address

    RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA., RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA. colin.wu2@nih.gov.,
    1. Year: 2022
  2. Journal: Methods in Molecular Biology (Clifton, N.J.)
    1. 2428
    2. Pages: 173-186
  4. Type of Article: Article
  1. 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. © 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

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External Sources

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  2. DOI: 10.1007/978-1-0716-1975-9_11
  3. PMID: 35171480

Library Notes

  1. Fiscal Year: FY2021-2022
  2. Group/Lab/Department: RNA Biology Laboratory
NCI at Frederick

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