Department of Chemistry, Emory University, Atlanta, Georgia, USA; Graduate Program in Biochemistry, Cell and Developmental Biology, Emory University, Atlanta, Georgia, USA.
Department of Chemistry, Emory University, Atlanta, Georgia, USA.
J Biol Chem. 2024 Oct;300(10):107743. doi: 10.1016/j.jbc.2024.107743. Epub 2024 Sep 1.
Translation initiation is a highly regulated, multi-step process that is critical for efficient and accurate protein synthesis. In bacteria, initiation begins when mRNA, initiation factors, and a dedicated initiator fMet-tRNA bind the small (30S) ribosomal subunit. Specific binding of fMet-tRNA in the peptidyl (P) site is mediated by the inspection of the fMet moiety by initiation factor IF2 and of three conserved G-C base pairs in the tRNA anticodon stem by the 30S head domain. Tandem A-minor interactions form between 16S ribosomal RNA nucleotides A1339 and G1338 and tRNA base pairs G30-C40 and G29-C41, respectively. Swapping the G30-C40 pair of tRNA with C-G (called tRNA M1) reduces discrimination against the noncanonical start codon CUG in vitro, suggesting crosstalk between the gripping of the anticodon stem and recognition of the start codon. Here, we solved electron cryomicroscopy structures of Escherichia coli 70S initiation complexes containing the fMet-tRNA M1 variant paired to the noncanonical CUG start codon, in the presence or absence of IF2 and the non-hydrolyzable GTP analog GDPCP, alongside structures of 70S initiation complexes containing this tRNA variant paired to the canonical bacterial start codons AUG, GUG, and UUG. We find that the M1 mutation weakens A-minor interactions between tRNA and 16S nucleotides A1339 and G1338, with IF2 strengthening the interaction of G1338 with the tRNA minor groove. These structures suggest how even slight changes to the recognition of the fMet-tRNA anticodon stem by the ribosome can impact the start codon selection.
翻译起始是一个高度调控的多步骤过程，对蛋白质合成的高效和准确性至关重要。在细菌中，起始始于 mRNA、起始因子和专用起始 fMet-tRNA 结合小（30S）核糖体亚基时。fMet-tRNA 在肽酰（P）位的特异性结合由起始因子 IF2 检查 fMet 部分和 30S 头部结构域检查 tRNA 反密码子茎中的三个保守 G-C 碱基对介导。16S 核糖体 RNA 核苷酸 A1339 和 G1338 与 tRNA 碱基对 G30-C40 和 G29-C41 之间分别形成串联 A- 小键相互作用。tRNA 与 G30-C40 对的交换与 C-G（称为 tRNA M1）降低了体外对非典型起始密码子 CUG 的区分，表明反密码子茎的夹持与起始密码子的识别之间存在串扰。在这里，我们解决了含有非典型 CUG 起始密码子的 fMet-tRNA M1 变体与 IF2 和非水解 GTP 类似物 GDPCP 配对的大肠杆菌 70S 起始复合物的电子 cryomicroscopy 结构，以及含有该 tRNA 变体与典型细菌起始密码子 AUG、GUG 和 UUG 配对的 70S 起始复合物的结构。我们发现 M1 突变削弱了 tRNA 与 16S 核苷酸 A1339 和 G1338 之间的 A- 小键相互作用，IF2 增强了 G1338 与 tRNA 小沟的相互作用。这些结构表明，核糖体对 fMet-tRNA 反密码子茎的识别的微小变化如何影响起始密码子的选择。
