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.
Department of Biochemistry, The University of Western Ontario, London, Canada.
Susanna
S
George
Department of Biochemistry, The University of Western Ontario, London, Canada.
Jacob D
JD
Aguirre
Department of Biochemistry, The University of Western Ontario, London, Canada.
Donald E
DE
Spratt
Department of Biochemistry, The University of Western Ontario, London, Canada.
Yumin
Y
Bi
Department of Biochemistry, The University of Western Ontario, London, Canada.
Madeline
M
Jeffery
Department of Biochemistry, The University of Western Ontario, London, Canada.|Department of Chemistry, The University of Western Ontario, London, Canada.
Gary S
GS
Shaw
Department of Biochemistry, The University of Western Ontario, London, Canada.|Department of Chemistry, The University of Western Ontario, London, Canada.
Patrick
P
O'Donoghue
D018388:Codon, Terminator; D004926:Escherichia coli / Q000235:genetics; D016368:Frameshift Mutation; D006801:Humans; D010768:Phosphoserine / Q000378:metabolism*; D014176:Protein Biosynthesis; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism; D054875:Ubiquitination
D018388:Codon, Terminator; D025801:Ubiquitin; D010768:Phosphoserine; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein
Parkinson's disease; codon skipping; neurodegeneration; phosphoseryl-tRNA synthetase (SepRS); synthetic biology