Efficiency of protein synthesis on the ribosome is strongly affected by the amino acid composition of the assembled amino acid chain. Challenging sequences include proline-rich motifs as well as highly positively and negatively charged amino acid stretches. Members of the F subfamily of ABC ATPases (ABCFs) have been long hypothesised to promote translation of such problematic motifs. In this study we have applied genetics and reporter-based assays to characterise the four housekeeping ABCF ATPases of Bacillus subtilis: YdiF, YfmM, YfmR/Uup and YkpA/YbiT. We show that YfmR cooperates with the translation factor EF-P that promotes translation of Pro-rich motifs. Simultaneous loss of both YfmR and EF-P results in a dramatic growth defect. Surprisingly, this growth defect can be largely suppressed though overexpression of an EF-P variant lacking the otherwise crucial 5-amino-pentanolylated residue K32. Using in vivo reporter assays, we show that overexpression of YfmR can alleviate ribosomal stalling on Asp-Pro motifs. Finally, we demonstrate that YkpA/YbiT promotes translation of positively and negatively charged motifs but is inactive in resolving ribosomal stalls on proline-rich stretches. Collectively, our results provide insights into the function of ABCF translation factors in modulating protein synthesis in B. subtilis.

中文翻译：

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通过 EF-P 和 ABCF ATP 酶 YfmR 和 YkpA/YbiT 解决核糖体停滞


核糖体上蛋白质合成的效率很大程度上受到组装氨基酸链的氨基酸组成的影响。具有挑战性的序列包括富含脯氨酸的基序以及高度带正电和带负电的氨基酸序列。长期以来，人们一直假设 ABC ATP 酶 (ABCF) 的 F 亚家族成员能够促进此类有问题的基序的翻译。在这项研究中，我们应用遗传学和基于报告基因的测定来表征枯草芽孢杆菌的四种管家 ABCF ATP 酶：YdiF、YfmM、YfmR/Uup 和 YkpA/YbiT。我们发现 YfmR 与翻译因子 EF-P 合作，促进富含 Pro 的基序的翻译。 YfmR 和 EF-P 同时丢失会导致严重的生长缺陷。令人惊讶的是，通过过度表达缺少关键的 5-氨基戊酰化残基 K32 的 EF-P 变体，可以在很大程度上抑制这种生长缺陷。使用体内报告基因检测，我们发现 YfmR 的过度表达可以减轻 Asp-Pro 基序上的核糖体停滞。最后，我们证明 YkpA/YbiT 促进带正电荷和带负电荷的基序的翻译，但在解决富含脯氨酸的片段上的核糖体失速方面没有活性。总的来说，我们的结果提供了对 ABCF 翻译因子在调节枯草芽孢杆菌蛋白质合成中的功能的见解。