Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA.
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
Nucleic Acids Res. 2021 Sep 20;49(16):9444-9458. doi: 10.1093/nar/gkab655.
The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg2+-dependent cleavage of the 5' leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250-500 mM Mg2+ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg2+ requirement to ∼10-20 mM and improves the rate and fidelity of cleavage. To understand the Mg2+- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPRC domain and Cy5-RPRS domain), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg2+-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg2+ cooperativity. Our findings highlight how Mg2+ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.
古菌核糖核酸酶 P 的核蛋白（RNP）形式由一个催化 RNA 和五个蛋白辅因子组成。为了催化 Mg2+依赖性地切割前 tRNA 的 5'前导序列，核糖核酸酶 P RNA（RPR）的催化（C）和特异性（S）结构域合作识别前 tRNA 的不同部分。虽然在没有核糖核酸酶 P 蛋白（RPPs）的情况下，约 250-500mM 的 Mg2+可使古菌 RPR 具有活性，但添加所有 RPPs 可将 Mg2+的需求降低至约 10-20mM，并提高切割的速度和保真度。为了理解增加活性的 Mg2+和 RPP 依赖性结构变化，我们使用前 tRNA 切割和整体 FRET 测定法来表征 Pyrococcus furiosus（Pfu）RPR 中的结构域间相互作用，无论是单独存在还是与 RPPs±前 tRNA 一起存在。在用双链标记 RPR（Cy3-RPRC 结构域和 Cy5-RPRS 结构域）进行拼接连接后，我们使用天然质谱法来验证最终产物。我们发现 FRET 与活性密切相关，Pfu RPR 和核糖核酸酶 P 全酶（RPR+5 RPPs）通过不同的 Mg2+依赖性途径趋同于相似的功能状态，并且全酶中前 tRNA 的结合会影响 Mg2+协同性。我们的研究结果突出了多亚基 RNP 中的 Mg2+和蛋白质如何共同促进 RNA 构象在动态集合中获得功能增益。
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