Generation of phospho-ubiquitin variants by orthogonal translation reveals codon skipping

FEBS Lett. 2016 May;590(10):1530-42. doi: 10.1002/1873-3468.12182. Epub 2016 May 4.

Abstract

The activity of the Parkinson's disease-linked E3 ligase parkin is stimulated by phosphorylation at ubiquitin Ser65 (pUb(S65) ). The role of other ubiquitin phospho-sites and their kinases are unknown. We produced pUb variants (pS7, pS12, pS20, pS57, pS65) by genetically encoding phosphoserine with the UAG codon. In release factor-deficient Escherichia coli (ΔRF1), intended to enhance UAG read-through, we discovered ubiquitin variants lacking the UAG-encoded residue, demonstrating previously undocumented +3 frame shifting. We successfully purified each pUb variant from mistranslated products. While pUb(S20) failed to stimulate parkin, parkin was partially active with pUb(S12) . We observed significant ubiquitination when pUb(S65) was the sole substrate.

Keywords: Parkinson's disease; codon skipping; neurodegeneration; phosphoseryl-tRNA synthetase (SepRS); synthetic biology.

Publication types

  • Letter

MeSH terms

  • Codon, Terminator*
  • Escherichia coli / genetics
  • Frameshift Mutation
  • Humans
  • Phosphoserine / metabolism*
  • Protein Biosynthesis
  • Ubiquitin / metabolism*
  • Ubiquitin-Protein Ligases / chemistry
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitination

Substances

  • Codon, Terminator
  • Ubiquitin
  • Phosphoserine
  • Ubiquitin-Protein Ligases
  • parkin protein