Translational regulation in the ribosome tunnel

我们一直在研究SecA的表达调控，SecA是一种细胞质atp酶，在大肠杆菌中通过SecYEG复合体的蛋白质易位中起主要作用。SecA的表达在翻译水平上由secM基因调控，secM基因位于SecA的上游，位于同一操纵子中，并编码分泌前蛋白。SecM含有一个17个氨基酸的c端序列基序（150FXXXXWIXXXXGIRAGP166），通常会引起短暂的翻译中断。而在分泌应激条件下，SecM的膜靶向性受到抑制。这种靶向缺陷通过同时改变secM-secA mRNA的结构，延长了翻译阻滞并增加了SecA的合成。翻译阻滞需要识别核糖体隧道内的SecM c端基序，但识别机制尚不清楚。虽然该基序的单一突变会损害识别，但我们发现可以通过重塑SecM c端来产生新的诱导阻滞的肽。我们发现R163是必不可少的，但在数量、位置和侧链化学上不同的侧翼残基在翻译阻滞中起着重要的次要作用。观察到单个SecM变体显示出与核糖体蛋白交联的独特模式，这表明每个肽在隧道内采用独特的构象。基于我们的研究结果，我们提出当侧翼残基指定的肽构象将R163移动到精确的隧道内位置时，发生翻译阻滞。我们的数据表明，翻译阻滞源于SecM和核糖体通道之间的广泛交流，并有助于解释在细菌、真菌和高等真核生物中发现的诱导阻滞肽的惊人多样性。

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 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. We have also 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. In a recent effort to identify novel translation arrest phenomena in E. coli, we compared the proteome of a wild-type strain to a strain that has a mutation in the ribosomal L22 subunit that appears to exert a broad inhibitory effect on peptide-mediated translation arrest. By impairing SecM-mediated translation arrest, the mutation reduces the steady-state level of SecA. While the mutation does not affect the level of most proteins, it dramatically reduces the level of antigen 43 (Ag43), a secreted virulence factor that promotes biofilm formation. We found, however, that the decrease in Ag43 concentration was an indirect effect of the reduction in secA expression and was not due to a defect in the recognition of a novel translation arrest-inducing peptide. Our results suggest that peptide-mediated translation arrest is used relatively infrequently to regulate gene expression in E. coli.