Quantifying the Binding of Fluorescently Labeled Guanine Nucleotides and Initiator tRNA to Eukaryotic Translation Initiation Factor 2

Martin D. Jennings, Graham D. Pavitt

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

The translation initiation factor eIF2 is critical for protein synthesis initiation, and its regulation is central to the integrated stress response (ISR). eIF2 is a G protein, and the activity is regulated by its GDP or GTP-binding status, such that only GTP-bound eIF2 has high affinity for initiator methionyl tRNA. In the ISR, regulatory signaling reduces the availability of eIF2-GTP and so downregulates protein synthesis initiation in cells. Fluorescence spectroscopy can be used as an analytical tool to study protein–ligand interactions in vitro. Here we describe methods to purify eIF2 and assays of its activity, employing analogs of GDP, GTP, and methionyl initiator tRNA ligands to accurately measure their binding affinities.

Original languageEnglish
Title of host publicationThe Integrated Stress Response
Subtitle of host publicationMethods and Protocols
EditorsDaniel Matějů, Jeffrey A. Chao
Place of PublicationNew York
PublisherHumana Press, Inc
Pages89-99
Number of pages11
ISBN (Electronic)9781071619759
ISBN (Print)9781071619742, 9781071619773
DOIs
Publication statusPublished - 17 Feb 2022

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
Volume2428
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • fluorescence spectroscopy
  • GDP
  • GTP
  • Met–tRNAi
  • translation initiation
  • eIF2

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