Optimized protocol for quantifying 5' UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting

STAR Protoc. 2022 Dec 16;3(4):101862. doi: 10.1016/j.xpro.2022.101862. Epub 2022 Nov 17.

Abstract

Direct analysis of ribosome targeting (DART) allows investigators to measure the translation initiation potential of thousands of RNAs in parallel. Here, we describe an optimized protocol for generating active translation extract from S. cerevisiae, followed by in vitro translation, purification of ribosome-bound RNAs, and subsequent library preparation and sequencing. This protocol can be applied to a variety of cell types and will enable high-throughput interrogation of translational determinants. For complete details on the use and execution of this protocol, please refer to Niederer et al. (2022).1.

Keywords: Gene Expression; Molecular Biology; Sequencing; Systems biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5' Untranslated Regions / genetics
  • Protein Biosynthesis* / genetics
  • RNA, Messenger / metabolism
  • RNA, Ribosomal / metabolism
  • Ribosomes / genetics
  • Ribosomes / metabolism
  • Saccharomyces cerevisiae* / genetics
  • Saccharomyces cerevisiae* / metabolism

Substances

  • 5' Untranslated Regions
  • RNA, Messenger
  • RNA, Ribosomal