Department of Physics and Physical Oceanography, Memorial University of Newfoundland, St Johns, Newfoundland, Canada.
Proteins. 2021 Mar;89(3):289-300. doi: 10.1002/prot.26014. Epub 2020 Oct 10.
RfaH is a compact two-domain bacterial transcription factor that functions both as a regulator of transcription and an enhancer of translation. Underpinning the dual functional roles of RfaH is a partial but dramatic fold switch, which completely transforms the ~50-amino acid C-terminal domain (CTD) from an all-α state to an all-β state. The fold switch of the CTD occurs when RfaH binds to RNA polymerase (RNAP), however, the details of how this structural transformation is triggered is not well understood. Here we use all-atom Monte Carlo simulations to characterize structural fluctuations and mechanical stability properties of the full-length RfaH and the CTD as an isolated fragment. In agreement with experiments, we find that interdomain contacts are crucial for maintaining a stable, all-α CTD in free RfaH. To probe mechanical properties, we use pulling simulations to measure the work required to inflict local deformations at different positions along the chain. The resulting mechanical stability profile reveals that free RfaH can be divided into a "rigid" part and a "soft" part, with a boundary that nearly coincides with the boundary between the two domains. We discuss the potential role of this feature for how fold switching may be triggered by interaction with RNAP.
RfaH 是一种紧凑的双结构域细菌转录因子，既能作为转录的调节剂，又能增强翻译。RfaH 的双重功能作用的基础是部分但显著的折叠开关，它完全将约 50 个氨基酸的 C 末端结构域（CTD）从全α状态转变为全β状态。当 RfaH 与 RNA 聚合酶（RNAP）结合时，CTD 的折叠开关会发生，但这种结构转换是如何触发的细节尚不清楚。在这里，我们使用全原子蒙特卡罗模拟来描述全长 RfaH 和作为分离片段的 CTD 的结构波动和机械稳定性特性。与实验一致，我们发现结构域间的接触对于在游离 RfaH 中维持稳定的全α CTD 至关重要。为了探测机械特性，我们使用拉伸模拟来测量在链上不同位置施加局部变形所需的功。所得的机械稳定性谱表明，游离 RfaH 可以分为“刚性”部分和“柔软”部分，其边界几乎与两个结构域的边界重合。我们讨论了这一特征在与 RNAP 相互作用时如何引发折叠开关的潜在作用。
