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

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.