Biological functions of the ribosomal exit tunnel

SecA是一种三磷酸腺苷酶，通过SecYEG易位驱动蛋白质穿过细菌细胞质膜输出。在大肠杆菌中，secA基因之前有一个开放的阅读框，称为基因X或secM（分泌监测），它包含一个带有secA的操纵子。我们对基因X产物的表征表明，它包含一个与核糖体出口通道相互作用以阻止翻译延伸的“捕获序列”。我们发现，在蛋白质分泌受损的条件下（如低温），延长阻滞对于SecA的基础水平表达以及其上调是必不可少的。此外，我们的研究结果表明，SecM促进了新合成的SecA分子的功能化，可能是通过将其生物合成定位到膜/转座子附近，我们称之为“顺式伴侣”的功能。因此，SecM仅在其新生的核糖体拴系状态下起作用，并且包含一个在核糖体翻译中不能被延长的序列，除非核糖体外的n端区域与Sec机制相互作用。我们利用纯化的翻译成分在体外翻译系统中研究了阻止延伸的机制，发现SecM的特性包括其Pro166密码子指定位于核糖体a位的脯氨酸- trna，该脯氨酸- trna作为阻止延伸的效应物而不被纳入多肽链。

SecA is an ATPase that drives protein export across the bacterial cytoplasmic membrane, through the SecYEG translocon. In E. coli the gene secA is preceded by an open reading frame called gene X or secM (secretion monitor), which comprises an operon with secA. Our characterization of the gene X product revealed that it contains an "arrest sequence" that interacts with the exit tunnel of the ribosome to halt translation elongation. We showed that the elongation arrest is essential for the basal level expression of SecA as well as for its up-regulation under the conditions (such as low temperture) of impaired protein secretion. Moreover, our results suggest that SecM falicilitates the functionalization of newly synthesized SecA molecules, presumably by localizing its biosynthesis to the vicinity of the membrane/ translocon, a function we refer to "cis-chaperone". Thus, SecM functions exclusively in its nascent ribosome-tethered state and contains a sequence that cannot be elongated in ribosomal translation unless the N-terminal region outside the ribosome interacts with the Sec machinery. We investigated the mechanism of elongation arrest using in vitro translation system with purified translation components and revealed that the SecM peculiarity includes the fact that its Pro166 codon specifies ribosomal A-site located prolyl-tRNA that functions as an effector of the elongation arrest without being incorporated into the polypeptide chain.