Generation of phospho-ubiquitin variants by orthogonal translation reveals codon skipping
Generation of phospho-ubiquitin variants by orthogonal translation reveals codon skipping
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DOI：
10.1002/1873-3468.12182
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发表时间：
2016-05-01
期刊：
影响因子：
3.5
通讯作者：
O'Donoghue, Patrick
中科院分区：
文献类型：
--
作者：
George, Susanna;Aguirre, Jacob D.;O'Donoghue, Patrick
帕金森病相关的E3连接酶parkin的活性被泛素Ser65(PUB)的磷酸化所刺激(S65)。其他泛素磷酸化位点和它们的激酶的作用尚不清楚。我们用UAG密码子对磷酸丝氨酸进行遗传编码，产生了PUB变体(PS7、PS12、pS20、pS57、pS65)。在释放因子缺陷型大肠杆菌(RF1)中，我们发现了缺乏UAG编码残基的泛素变体，显示了以前未见文献报道的+3帧移位。我们成功地从误译的产品中提纯了每一种酒吧变体。PUB(S20)不能刺激PARKIN，PUB(S12)PARKIN部分激活。当PUB(S65)为唯一底物时，我们观察到显著的泛素化。
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 (RF1), 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.