Translational regulation in the ribosome tunnel

我们一直在研究SecA的表达调控，这是一种细胞质ATPase，在大肠杆菌中通过SecYEG复合体转运蛋白质起着重要作用。SECM在翻译水平上调节SECA的表达，SECM是一种位于SECA上游的基因，位于同一操纵子的上游，编码一种记忆前蛋白。SECM含有一个17个氨基酸的C-末端序列基序(150FXXXXWIXXXXGIRAGP166)，通常会导致瞬时翻译停滞。然而，在分泌胁迫条件下，SECM的膜靶向性受到抑制。这种靶向缺陷通过改变SECM-SECA mRNA的结构延长了翻译停滞，并增加了SECA的合成。翻译抑制需要识别核糖体隧道内的SECM C-末端基序，但识别机制尚不清楚。虽然基序中的单个突变会削弱识别能力，但我们发现，通过SECM C-末端的重塑可以产生新的停滞诱导肽。我们发现R163是必不可少的，但侧翼残基在翻译抑制中起着重要的辅助作用，这些残基在数量、位置和侧链化学上都不同。观察到单个SECM变体显示出与核糖体蛋白不同的交联模式，这表明每个肽在隧道内都采用了一种独特的构象。根据我们的结果，我们认为当侧翼残基指定的多肽构象将R163移动到准确的隧道内位置时，翻译停滞发生。我们的数据表明，翻译停滞是SECM和核糖体隧道之间广泛沟通的结果，并有助于解释在细菌、真菌和高等真核生物中发现的停滞诱导肽的惊人多样性。 最近，我们发现SECM信号肽在这一调控过程中起着至关重要的作用，它作为一个分子计时器来协调膜靶向和阻止基序的合成。我们发现，改变靶向动力学的信号肽突变以及改变SECM信号肽和停滞基序之间距离的插入或缺失会扰乱适当调节SecA合成所需的停滞开始和释放之间的平衡。此外，我们发现核糖体和SECM阻止基序之间的相互作用的强度被校准，以确保在膜靶向时阻止的释放。我们的结果强烈表明，SECM蛋白的几个独特特征是由于核糖体和SEC机制施加的限制而进化的。

We have been investigating the regulation of expression of SecA, a cytoplasmic ATPase that plays a major role in the translocation of proteins through the SecYEG complex in E. coli. SecA expression is regulated at the translational level by secM, a gene that resides immediately upstream of secA in the same operon and that encodes a presecretory protein. SecM contains a 17 amino acid C-terminal sequence motif (150FXXXXWIXXXXGIRAGP166) that normally induces a transient translation arrest. Under conditions of secretion stress, however, the membrane targeting of SecM is inhibited. This targeting defect prolongs the translation arrest and increases SecA synthesis by concomitantly altering the structure of the secM-secA mRNA. Translation arrest requires the recognition of the SecM C-terminal motif inside the ribosome tunnel, but the mechanism of recognition is unknown. While single mutations in the motif impair recognition, we found that novel arrest-inducing peptides can be created through remodeling of the SecM C-terminus. We found that R163 is indispensable, but that flanking residues that vary in number, position, and side chain chemistry play an important secondary role in translation arrest. The observation that individual SecM variants show a distinct pattern of crosslinking to ribosomal proteins suggests that each peptide adopts a unique conformation inside the tunnel. Based on our results, we propose that translation arrest occurs when the peptide conformation specified by flanking residues moves R163 into a precise intra-tunnel location. Our data indicate that translation arrest results from extensive communication between SecM and the ribosome tunnel and help explain the striking diversity of arrest-inducing peptides found in bacteria, fungi and higher eukaryotes. Recently, we found that the SecM signal peptide plays an essential role in this regulatory process by acting as a molecular timer that coordinates membrane targeting with the synthesis of the arrest motif. We found that signal peptide mutations that alter targeting kinetics and insertions or deletions that change the distance between the SecM signal peptide and the arrest motif perturb the balance between the onset and release of arrest that is required to regulate SecA synthesis properly. Furthermore, we found that the strength of the interaction between the ribosome and the SecM arrest motif is calibrated to ensure the release of arrest upon membrane targeting. Our results strongly suggest that several distinctive features of the SecM protein evolved as a consequence of constraints imposed by the ribosome and the Sec machinery.