Purification and Use of tRNA for Enzymatic Post-translational Addition of Amino Acids to Proteins

STAR Protoc. 2020 Dec 9;1(3):100207. doi: 10.1016/j.xpro.2020.100207. eCollection 2020 Dec 18.

Abstract

Post-translational addition of amino acids to proteins by enzymes using aminoacyl-tRNA is an emerging regulatory mechanism. Examples include Arg transfer in eukaryotes, Leu/Phe transfer in bacteria, and tRNA-synthetase-mediated addition of amino acids to Lys side chains. Here, we present a method of purification and use of tRNA for such reactions, focusing on tRNAArg and its use for arginylation. This method can also be used for other tRNA-mediated reactions. For complete details on the use and execution of this protocol, please refer to Avcilar-Kucukgoze et al. (2020).

Keywords: Molecular Biology; Molecular/Chemical Probes; Protein Biochemistry.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acids / metabolism
  • Amino Acyl-tRNA Synthetases / chemistry
  • Arginine / chemistry
  • Enzyme Assays / methods*
  • Protein Processing, Post-Translational / physiology*
  • RNA, Transfer / genetics
  • RNA, Transfer / isolation & purification*
  • RNA, Transfer / metabolism
  • RNA, Transfer / physiology
  • RNA, Transfer, Amino Acyl / metabolism
  • Transcription, Genetic / physiology

Substances

  • Amino Acids
  • RNA, Transfer, Amino Acyl
  • RNA, Transfer
  • Arginine
  • Amino Acyl-tRNA Synthetases