Department of Applied Physics, Stanford University, Stanford, CA 94305.
Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305.
Proc Natl Acad Sci U S A. 2017 Dec 26;114(52):13691-13696. doi: 10.1073/pnas.1719592115. Epub 2017 Dec 11.
During protein synthesis, the ribosome simultaneously binds up to three different transfer RNA (tRNA) molecules. Among the three tRNA binding sites, the regulatory role of the exit (E) site, where deacylated tRNA spontaneously dissociates from the translational complex, has remained elusive. Here we use two donor-quencher pairs to observe and correlate both the conformation of ribosomes and tRNAs as well as tRNA occupancy. Our results reveal a partially rotated state of the ribosome wherein all three tRNA sites are occupied during translation elongation. The appearance and lifetime of this state depend on the E-site tRNA dissociation kinetics, which may vary among tRNA species and depends on temperature and ionic strength. The 3-tRNA partially rotated state is not a proper substrate for elongation factor G (EF-G), thus inhibiting translocation until the E-site tRNA dissociates. Our result presents two parallel kinetic pathways during translation elongation, underscoring the ability of E-site codons to modulate the dynamics of protein synthesis.
在蛋白质合成过程中，核糖体同时结合多达三个不同的转移 RNA（tRNA）分子。在三个 tRNA 结合位点中，脱酰基 tRNA 自发从翻译复合物中解离的出口（E）位点的调节作用仍然难以捉摸。在这里，我们使用两对供体-猝灭剂对来观察和关联核糖体和 tRNA 的构象以及 tRNA 的占有率。我们的结果揭示了核糖体的部分旋转状态，其中在翻译延伸过程中三个 tRNA 位点都被占据。这种状态的出现和寿命取决于 E 位 tRNA 的解离动力学，这可能因 tRNA 种类而异，并取决于温度和离子强度。3-tRNA 部分旋转状态不是延伸因子 G（EF-G）的合适底物，因此在 E 位 tRNA 解离之前抑制易位。我们的结果在翻译延伸过程中呈现出两种平行的动力学途径，强调了 E 位密码子调节蛋白质合成动力学的能力。
