In most bacterial type A RNase P RNAs (P RNAs), two major loop-helix tertiary contacts (L8–P4 and L18–P8) help to orient the two independently folding S- and C-domains for concerted recognition of precursor tRNA substrates. Here, we analyze the effects of mutations in these tertiary contacts in P RNAs from three different species: (i) the psychrophilic bacterium Pseudoalteromonas translucida (Ptr), (ii) the mesophilic radiation-resistant bacterium Deinococcus radiodurans (Dra), and (iii) the thermophilic bacterium Thermus thermophilus (Tth). We show by UV melting experiments that simultaneous disruption of these two interdomain contacts has a stabilizing effect on all three P RNAs. This can be inferred from reduced RNA unfolding at lower temperatures and a more concerted unfolding at higher temperatures. Thus, when the two domains tightly interact via the tertiary contacts, one domain facilitates structural transitions in the other. P RNA mutants with disrupted interdomain contacts showed severe kinetic defects that were most pronounced upon simultaneous disruption of the L8–P4 and L18–P8 contacts. At 37°C, the mildest effects were observed for the thermostable Tth RNA. A third interdomain contact, L9–P1, makes only a minor contribution to P RNA tertiary folding. Furthermore, D. radiodurans RNase P RNA forms an additional pseudoknot structure between the P9 and P12 of its S-domain. This interaction was found to be particularly crucial for RNase P holoenzyme activity at near-physiological Mg²⁺ concentrations (2 mM). We further analyzed an exceptionally stable folding trap of the G,C-rich Tth P RNA.

中文翻译：

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来自嗜冷、耐温/抗辐射和嗜热细菌的 RNase P RNA 三级接触和折叠的比较研究

在大多数细菌 A 型 RNase P RNA (P RNA) 中，两个主要的环螺旋三级接触（L8-P4 和 L18-P8）有助于定位两个独立折叠的 S 和 C 结构域，以协同识别前体 tRNA 底物。在这里，我们分析了来自三个不同物种的 P RNA 中这些三级接触中突变的影响：(i) 嗜冷细菌Pseudoalteromonas translucida ( Ptr )，(ii) 嗜温耐辐射细菌Deinococcus radiodurans ( Dra ) ，以及 (iii) ) 嗜热细菌嗜热栖热菌( Tth）。我们通过 UV 熔解实验表明，这两个域间接触的同时破坏对所有三个 P RNA 都有稳定作用。这可以从在较低温度下减少的 RNA 去折叠和在较高温度下更加协调的展开来推断。因此，当两个域通过三级接触紧密相互作用时，一个域促进另一个域的结构转变。具有破坏的域间接触的 P RNA 突变体显示出严重的动力学缺陷，这在 L8-P4 和 L18-P8 接触同时破坏时最为明显。在 37°C 时，观察到对热稳定的Tth RNA 的影响最温和。第三个域间接触 L9-P1 对 P RNA 三级折叠的贡献很小。此外，D. radioduransRNase P RNA 在其 S 域的 P9 和 P12 之间形成一个额外的假结结构。发现这种相互作用对于接近生理学 Mg ²⁺浓度 (2 mM) 的RNase P 全酶活性特别重要。我们进一步分析了富含 G,C 的Tth P RNA的异常稳定的折叠陷阱。