Mercier Evan, Holtkamp Wolf, Rodnina Marina V, Wintermeyer Wolfgang
Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen 37077, Germany.
Nucleic Acids Res. 2017 Nov 16;45(20):11858-11866. doi: 10.1093/nar/gkx888.
The bacterial signal recognition particle (SRP) is part of the machinery that targets ribosomes synthesizing membrane proteins to membrane-embedded translocons co-translationally. Recognition of nascent membrane proteins occurs by virtue of a hydrophobic signal-anchor sequence (SAS) contained in the nascent chain, usually at the N terminus. Here we use fluorescence-based stopped-flow to monitor SRP-ribosome interactions with actively translating ribosomes while an SRP substrate is synthesized and emerges from the peptide exit tunnel. The kinetic analysis reveals that, at cellular concentrations of ribosomes and SRP, SRP rapidly binds to translating ribosomes prior to the emergence of an SAS and forms an initial complex that rapidly rearranges to a more stable engaged complex. When the growing peptide reaches a length of ∼50 amino acids and the SAS is partially exposed, SRP undergoes another conformational change which further stabilizes the complex and initiates targeting of the translating ribosome to the translocon. These results provide a reconciled view on the timing of high-affinity targeting complex formation, while emphasizing the existence of preceding SRP recruitment steps under conditions of ongoing translation.
细菌信号识别颗粒（SRP）是将合成膜蛋白的核糖体共翻译靶向膜嵌入转运体的机制的一部分。新生膜蛋白的识别是通过新生链中通常位于N端的疏水信号锚定序列（SAS）实现的。在这里，我们使用基于荧光的停流技术来监测SRP-核糖体与正在进行翻译的核糖体的相互作用，同时合成一个SRP底物并使其从肽出口通道中出现。动力学分析表明，在核糖体和SRP的细胞浓度下，SRP在SAS出现之前迅速与正在翻译的核糖体结合，形成一个初始复合物，该复合物迅速重排为更稳定的结合复合物。当生长的肽达到约50个氨基酸的长度且SAS部分暴露时，SRP会发生另一种构象变化，这进一步稳定了复合物，并启动了将正在翻译的核糖体靶向转运体的过程。这些结果提供了关于高亲和力靶向复合物形成时间的协调观点，同时强调了在持续翻译条件下先前SRP招募步骤的存在。
