Global mapping of translation initiation sites by TIS profiling in budding yeast

STAR Protoc. 2020 Dec 30;2(1):100250. doi: 10.1016/j.xpro.2020.100250. eCollection 2021 Mar 19.

Abstract

Translation initiation site (TIS) profiling allows for the genome-wide identification of TISs in vivo by exclusively capturing mRNA fragments within ribosomes that have just completed translation initiation. It leverages translation inhibitors, such as harringtonine and lactimidomycin (LTM), that preferentially capture ribosomes at start codon positions, protecting TIS-derived mRNA fragments from nuclease digestion. Here, we describe a step-by-step protocol for TIS profiling in LTM-treated budding yeast that we developed to identify TISs and open reading frames in vegetative and meiotic cells. For complete details on the use and execution of this protocol, please refer to Eisenberg et al. (2020).

Keywords: Cell biology; Genomics; High-throughput screening; Model organisms; Molecular biology.

Publication types

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

MeSH terms

  • Codon, Initiator*
  • Open Reading Frames*
  • Peptide Chain Initiation, Translational*
  • Ribosomes / genetics
  • Ribosomes / metabolism*
  • Saccharomyces cerevisiae Proteins* / biosynthesis
  • Saccharomyces cerevisiae Proteins* / genetics
  • Saccharomyces cerevisiae* / genetics
  • Saccharomyces cerevisiae* / metabolism

Substances

  • Codon, Initiator
  • Saccharomyces cerevisiae Proteins