Department of Structural Biology, Stanford University, Stanford, CA 94305-5126, USA.
Program in Biophysics, Stanford University, Stanford, CA 94305-5126, USA.
Nucleic Acids Res. 2022 Oct 14;50(18):10201-10211. doi: 10.1093/nar/gkac576.
Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNAUTu1, a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNAUTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.
核糖体在接受来自同一生物体和其他生物体或生命领域的各种氨酰基-tRNA 底物方面具有很强的可塑性。这是核糖体的一个关键特征，它允许使用正交翻译系统来扩展遗传密码。这些正交翻译系统的优化通常涉及到 tRNA、氨酰基-tRNA 合成酶和非规范氨基酸之间的兼容性。随着我们扩展用于包括非规范结构的 tRNA 的多样性，出现了关于 tRNA 在核糖体上的适用性的问题。具体来说，我们使用单分子荧光法研究了 allo-tRNAUTu1 的核糖体翻译，allo-tRNAUTu1 是一种独特形状的（9/3）tRNA，用于特异性插入硒代半胱氨酸。通过这项技术，我们确定核糖体从 allo-tRNAUTu1 从 A 位到 P 位的易位中发生了解体。我们使用 cryo-EM 在核糖体上捕获 tRNA，发现了一种独特的三级相互作用，阻止了液体的易位。通过单个核苷酸突变，我们破坏了这种三级相互作用，从而缓解了翻译障碍。随着遗传密码扩展的持续多样化，我们的工作强调了一种针对特定 tRNA 的优化翻译的方法，因为它们在核糖体中移动。
