Translational regulation in the ribosome tunnel

我们一直在研究SecA的表达调控，SecA是一种胞质ATP酶，在E.杆菌编码SecA的基因位于编码SecM的基因的下游，并且两个基因形成单个操纵子。当分泌受损时，SecM的C-末端附近的17个氨基酸基序（150FXXXXWIXXXXGIRAGP166）诱导翻译停滞。该基序在核糖体隧道内被识别，但识别机制尚不清楚。虽然基序中的单个突变损害识别，但我们发现可以通过重塑SecM C-末端来创建新的抑制诱导肽。我们发现，R163是必不可少的，但侧翼残基的数量，位置和侧链化学变化发挥重要的次要作用，翻译逮捕。个别SecM变体显示与核糖体蛋白交联的独特模式的观察表明，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，翻译逮捕时发生的肽构象指定的侧翼残基移动R163到一个精确的隧道内的位置。我们的数据表明，翻译逮捕SecM和核糖体隧道之间的广泛沟通的结果，并帮助解释逮捕诱导肽在细菌，真菌和高等真核生物中发现的惊人的多样性。 我们还通过分析与大肠杆菌相关的异常长（55个氨基酸）的信号肽，意外地了解了核糖体隧道的功能。coli EspP蛋白。EspP信号肽含有25个残基的N-末端延伸（EspP1 - 25），我们显示其抑制信号肽被信号识别颗粒（SRP）识别。我们还发现EspP1 - 25与细胞质蛋白（MetE）的融合导致MetE聚集。两条证据表明，这两种效应都归因于核糖体隧道内EspP1 - 25的构象。首先，EspP1 - 25中的突变消除了其对蛋白质靶向和蛋白质折叠的影响，改变了短新生链与核糖体组分的交联。其次，L22中扭曲隧道的突变模仿了EspP1 - 25突变对蛋白质生物合成的影响。我们的研究结果提供了证据，核糖体隧道内的多肽的构象可以影响蛋白质折叠在生理条件下。

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.