Identification of endogenous SsrA-tagged proteins reveals tagging at positions corresponding to stop codons
Identification of endogenous SsrA-tagged proteins reveals tagging at positions corresponding to stop codons
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DOI：
10.1074/jbc.m103864200
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发表时间：
2001-07-27
影响因子：
4.8
通讯作者：
Sauer, RT
中科院分区：
文献类型：
--
作者：
Roche, ED;Sauer, RT
SsrA.SmpB质量控制系统将C-末端降解肽（AANDENYALAA）添加到停滞核糖体上的新生链，从而释放核糖体并确保标记蛋白的蛋白水解。使用具有标签序列AANDEHHHHHH的SsrA突变体来减缓降解并促进Ni 2 +-次氮基三乙酸亲和纯化。在二维凝胶上展示亲和纯化的大肠杆菌蛋白质显示了少量不同的SsrA-H-6标记蛋白质组，质谱鉴定了LaeI阻遏物、λ cI阻遏物、YbeL、GalE、RbsK和SlyD-kan（R）融合蛋白质。对于A阻遏物和YbeL，SsrA-H-6标签被添加在蛋白质的天然C末端之后，这表明当核糖体在这些基因的终止密码子处空闲时发生标签化。标记其他蛋白质的潜在原因包括来自下游阅读框翻译的干扰、稀有密码子和基因破坏。这些和以前的结果支持SsrA.SmpB系统在释放停滞的核糖体和指导这些受阻的蛋白质合成反应产物的降解中的广泛作用。
The SsrA.SmpB quality control system adds a C-terminal degradation peptide (AANDENYALAA) to nascent chains on stalled ribosomes, thereby freeing the ribosome and ensuring proteolysis of the tagged protein. An SsrA mutant with the tag sequence AANDEHHHHHH was used to slow degradation and facilitate, Ni2+-nitrilotriacetic acid affinity purification. Display of affinity-purified Escherichia coli proteins on two-dimensional gels revealed small quantities of a diverse set of SsrA-H-6-tagged proteins, and mass spectroscopy identified LaeI repressor, lambda cI repressor, YbeL, GalE, RbsK, and a SlyD-kan(R) fusion protein as members of this set. For A repressor and YbeL, the SsrA-H-6 tag was added after the natural C terminus of the protein, suggesting that tagging occurred while the ribosome idled at the termination codon of these genes. Potential causes of tagging for the other proteins include interference from translation of downstream reading frames, rare codons, and gene disruption. These and previous results support a broad role for the SsrA.SmpB system in freeing stalled ribosomes and in directing degradation of the products of these frustrated protein synthesis reactions.