Using SecM arrest sequence as a tool to isolate ribosome bound polypeptides.
Using SecM arrest sequence as a tool to isolate ribosome bound polypeptides.
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使用 SecM 阻滞序列作为工具来分离核糖体结合的多肽。
DOI：
10.3791/4027
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
A. Komar
中科院分区：
文献类型：
--
作者：
Sujata Jha;A. Komar
大量的研究表明，蛋白质在细胞中的折叠是一个共翻译过程。然而，多肽链在共翻译折叠过程中遵循的确切途径以实现其功能形式仍然是一个谜。为了理解这一过程并确定共翻译折叠中间体的确切构象，必须开发允许分离携带预定大小的新生链的RNC的技术，以允许其进一步的结构分析。SecM（分泌监测器）是170个氨基酸的E.在secM-secA操纵子中调节下游SecA（分泌驱动）ATP酶表达的大肠杆菌蛋白。Nakatogawa和Ito最初发现，SecM蛋白C-末端区域中的17个氨基酸长的序列（150-FSTPVWISQAQGIRAGP-166）足以且必须引起SecM在Gly 165处的延伸停滞，从而产生稳定结合到核糖体P-位点的肽基-甘氨酰-tRNA。更重要的是，发现该17个氨基酸长的序列可以融合到几乎任何全长和/或截短的蛋白质的C末端，从而允许产生携带预定大小的新生链的RNC。因此，当融合或插入靶蛋白时，SecM停滞序列会阻止多肽链延伸，并在大肠杆菌体内产生稳定的RNC。大肠杆菌细胞和体外无细胞系统。进一步利用蔗糖梯度离心分离RNC。分离的RNCs可用于分析共翻译折叠中间体的结构和功能特征。最近，这项技术已成功地用于深入了解几个核糖体结合新生链的结构。在这里，我们描述了牛γ-B晶体蛋白RNCs融合SecM和在体外翻译系统中产生的分离。
Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis. SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs. The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.
影响因子：
56.9
作者：
Ban, N;Nissen, P;Steitz, TA
通讯作者：
Steitz, TA