Ousalem Farès, Singh Shikha, Bailey Nevette A, Wong Kam-Ho, Zhu Lingwei, Neky Matthew J, Sibindi Cosmas, Fei Jingyi, Gonzalez Ruben L, Boël Grégory, Hunt John F
bioRxiv. 2023 Jun 12:2023.06.11.543863. doi: 10.1101/2023.06.11.543863.
Multiple paralogous ABCF ATPases are encoded in most genomes, but the physiological functions remain unknown for most of them. We herein compare the four K12 ABCFs - EttA, Uup, YbiT, and YheS - using assays previously employed to demonstrate EttA gates the first step of polypeptide elongation on the ribosome dependent on ATP/ADP ratio. A Δ knockout, like Δ , exhibits strongly reduced fitness when growth is restarted from long-term stationary phase, but neither Δ nor Δ exhibits this phenotype. All four proteins nonetheless functionally interact with ribosomes based on translation and single-molecule fluorescence resonance energy transfer experiments employing variants harboring glutamate-to-glutamine active-site mutations (EQ ) that trap them in the ATP-bound conformation. These variants all strongly stabilize the same global conformational state of a ribosomal elongation complex harboring deacylated tRNA in the P site. However, EQ -Uup uniquely exchanges on/off the ribosome on a second timescale, while EQ -YheS-bound ribosomes uniquely sample alternative global conformations. At sub-micromolar concentrations, EQ -EttA and EQ -YbiT fully inhibit translation of an mRNA encoding luciferase, while EQ -Uup and EQ -YheS only partially inhibit it at ~10-fold higher concentrations. Moreover, tripeptide synthesis reactions are not inhibited by EQ -Uup or EQ -YheS, while EQ -YbiT inhibits synthesis of both peptide bonds and EQ -EttA specifically traps ribosomes after synthesis of the first peptide bond. These results support the four ABCF paralogs all having different activities on translating ribosomes, and they suggest that there remains a substantial amount of functionally uncharacterized "dark matter" involved in mRNA translation.
大多数基因组中都编码了多个同源的ABCF ATP酶，但它们中大多数的生理功能仍不清楚。我们在此使用先前用于证明EttA在核糖体上多肽延伸的第一步依赖于ATP/ADP比率的检测方法，比较了四种K12 ABCF——EttA、Uup、YbiT和YheS。一个Δ敲除株，如Δ，当从长期稳定期重新开始生长时，其适应性会大幅降低，但Δ和Δ都不表现出这种表型。然而，基于使用携带谷氨酸到谷氨酰胺活性位点突变（EQ）的变体进行的翻译和单分子荧光共振能量转移实验，所有这四种蛋白质在功能上都与核糖体相互作用，这些变体将它们捕获在ATP结合构象中。这些变体都强烈稳定了在P位点含有脱酰基tRNA的核糖体延伸复合物的相同全局构象状态。然而，EQ -Uup在第二个时间尺度上独特地在核糖体上进行交换，而EQ -YheS结合的核糖体独特地采样替代的全局构象。在亚微摩尔浓度下，EQ -EttA和EQ -YbiT完全抑制编码荧光素酶的mRNA的翻译，而EQ -Uup和EQ -YheS仅在高约10倍的浓度下部分抑制它。此外，三肽合成反应不受EQ -Uup或EQ -YheS的抑制，而EQ -YbiT抑制两个肽键的合成，并且EQ -EttA在第一个肽键合成后特异性地捕获核糖体。这些结果支持这四种ABCF同源物在翻译核糖体上都具有不同的活性，并且它们表明在mRNA翻译中仍存在大量功能未表征的“暗物质”。
