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。编码SecA的基因位于编码SecM的基因的下游，这两个基因形成一个单一的操纵子。当分泌受损时，SecM c端附近的一个17个氨基酸基序（150FXXXXWIXXXXGIRAGP166）诱导翻译阻滞。该基序在核糖体隧道内被识别，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 the expression of SecA, a cytoplasmic ATPase that plays a major role in the translocation of proteins through the SecYEG complex in E. coli. The gene that encodes SecA resides immediately downstream of the gene that encodes SecM, and the two genes form a single operon. When secretion is impaired, a 17 amino acid motif (150FXXXXWIXXXXGIRAGP166) near the C-terminus of SecM induces a translation arrest. This motif is recognized 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 serendipitously obtained insight into the function of the ribosome tunnel through the analysis of an unusually long (55 amino acid) signal peptide associated with the E. coli EspP protein. The EspP signal peptide contains a 25 residue N-terminal extension (EspP1-25) that we showed inhibits signal peptide recognition by the signal recognition particle (SRP). We also found that the fusion of EspP1-25 to a cytoplasmic protein (MetE) causes MetE to aggregate. Two lines of evidence indicate that both of these effects are attributable to the conformation of EspP1-25 inside the ribosome tunnel. First, mutations in EspP1-25 that abolished its effects on protein targeting and protein folding altered the crosslinking of short nascent chains to ribosomal components. Second, a mutation in L22 that distorts the tunnel mimicked the effects of the EspP1-25 mutations on protein biogenesis. Our results provide evidence that the conformation of a polypeptide inside the ribosome tunnel can influence protein folding under physiological conditions.