Cui Da, Pestova Tatyana, Hellen Christopher, des Georges Amedee
Structural Biology Initiative, CUNY Advanced Science Research Center, City University of New York, New York, NY, 10031, USA.
Ph.D. Program in Biochemistry, The Graduate Center of the City University of New York, New York, NY, 10016, USA.
bioRxiv. 2025 Jul 14:2025.07.13.664561. doi: 10.1101/2025.07.13.664561.
The DExH-box helicase DHX29 plays a critical role in mammalian translation initiation. It is required for the scanning of mRNAs with complex 5'UTRs. Despite its importance, the detailed mechanism of DHX29's action has remained debated. Using structural models derived from AlphaFold and cryo-EM structure of DHX29 bound to the ribosomal 43S pre-initiation complex, we provide a revised structural framework that clarifies the interplay between DHX29, the 40S ribosomal subunit, and eIF3. Our findings suggest that the 40S subunit regulates DHX29's NTPase activity through an activation mechanism resembling the G-patch protein regulation of DEAH helicases. Moreover, our model supports a 3' to 5' translocase mechanism, in which DHX29 transiently pulls the mRNA opposite to the scanning direction, destabilizing stable stem-loops trapped in the mRNA channel and allowing scanning to resume. This structural analysis refines our understanding of DHX29's function and provides new hypotheses regarding its role in mRNA unwinding during scanning and start codon selection.
DExH盒解旋酶DHX29在哺乳动物翻译起始过程中发挥关键作用。它是扫描具有复杂5'非翻译区（5'UTR）的mRNA所必需的。尽管其很重要，但DHX29的详细作用机制仍存在争议。利用源自AlphaFold的结构模型以及与核糖体43S预起始复合物结合的DHX29的冷冻电镜结构，我们提供了一个经过修订的结构框架，阐明了DHX29、40S核糖体亚基和真核起始因子3（eIF3）之间的相互作用。我们的研究结果表明，40S亚基通过一种类似于DEAH解旋酶的G-结构域蛋白调控的激活机制来调节DHX29的NTPase活性。此外，我们的模型支持一种从3'到5'的转位酶机制，其中DHX29短暂地将mRNA向与扫描方向相反的方向拉动，使被困在mRNA通道中的稳定茎环结构不稳定，并允许扫描继续进行。这种结构分析完善了我们对DHX29功能的理解，并提供了关于其在扫描过程中mRNA解旋以及起始密码子选择中作用的新假设。
