Many bacteria harbor RNA-dependent nucleoside-triphosphatases of the DEAH/RHA family, whose molecular mechanisms and cellular functions are poorly understood. Here, we show that the Escherichia coli DEAH/RHA protein, HrpA, is an ATP-dependent 3 to 5′ RNA helicase and that the RNA helicase activity of HrpA influences bacterial survival under antibiotics treatment. Limited proteolysis, crystal structure analysis, and functional assays showed that HrpA contains an N-terminal DEAH/RHA helicase cassette preceded by a unique N-terminal domain and followed by a large C-terminal region that modulates the helicase activity. Structures of an expanded HrpA helicase cassette in the apo and RNA-bound states in combination with cross-linking/mass spectrometry revealed ratchet-like domain movements upon RNA engagement, much more pronounced than hitherto observed in related eukaryotic DEAH/RHA enzymes. Structure-based functional analyses delineated transient interdomain contact sites that support substrate loading and unwinding, suggesting that similar conformational changes support RNA translocation. Consistently, modeling studies showed that analogous dynamic intramolecular contacts are not possible in the related but helicase-inactive RNA-dependent nucleoside-triphosphatase, HrpB. Our results indicate that HrpA may be an interesting target to interfere with bacterial tolerance toward certain antibiotics and suggest possible interfering strategies.

许多细菌含有 DEAH/RHA 家族的 RNA 依赖性三磷酸核苷酶，但人们对其分子机制和细胞功能知之甚少。在这里，我们证明大肠杆菌DEAH/RHA 蛋白 HrpA 是一种 ATP 依赖性 3 至 5' RNA 解旋酶，并且 HrpA 的 RNA 解旋酶活性影响细菌在抗生素治疗下的存活。有限的蛋白水解、晶体结构分析和功能测定表明，HrpA 含有一个 N 端 DEAH/RHA 解旋酶盒，前面是一个独特的 N 端结构域，后面是一个调节解旋酶活性的大 C 端区域。与交联/质谱相结合，处于 apo 和 RNA 结合状态的扩展 HrpA 解旋酶盒的结构揭示了 RNA 接合时的棘轮状结构域运动，比迄今为止在相关真核 DEAH/RHA 酶中观察到的更为明显。基于结构的功能分析描绘了支持底物加载和解旋的瞬时域间接触位点，表明类似的构象变化支持 RNA 易位。模型研究一致表明，在相关但解旋酶失活的 RNA 依赖性核苷三磷酸酶 HrpB 中，类似的动态分子内接触是不可能的。我们的结果表明，HrpA 可能是干扰细菌对某些抗生素耐受性的一个有趣靶标，并提出了可能的干扰策略。