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. 2021 Sep 6;28(9):713–723. doi: 10.1038/s41594-021-00637-y

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 1 — a, SDS PAGE analysis of purified recombinant mtRNase P complex subunits. Lanes: M – protein molecular weight marker, 1 – co-purified Δ39-TRMT10C-SDR5C1 complex, 2 – Δ45-PRORP. Approximately 300 ng of purified proteins were loaded in lanes 1 and 2. b, Cleavage of pre-tRNA^Tyr by mtRNase P subunits and full mtRNase P complex. Lanes from left to right: RNA size marker, empty lane, only pre-tRNA, pre-tRNA with TRMT10C-SDR5C1, pre-tRNA with PRORP, and pre-tRNA with TRMT10C-SDR5C1 + PRORP. c, Effect of Mg²⁺ on pre-tRNA^Tyr cleavage by the mtRNase P complex. First three lanes from left: RNA size marker, empty lane, pre-tRNA without mtRNase P subunits or Mg²⁺. Subsequent lanes contain pre-tRNA + TRMT10C-SDR5C1 + PRORP and varying concentrations of Mg²⁺ as indicated. d, In-vitro reconstitution and purification of the mtRNase P complex by size exclusion chromatography (SEC) in Mg²⁺-depleted conditions. The SEC chromatogram between 0.80 and 2.00 mL retention volumes is shown. Eluate fractions between 0.85 and 1.55 mL were analyzed by SDS PAGE to assess protein content; SEC eluate fractions between 0.85 and 1.30 mL were additionally analyzed by Urea PAGE to assess RNA content. Fractions within the gray dashed rectangle (1.00–1.05 mL) were used for single particle analysis by cryo-EM. Additional replicates for panels b and c, along with uncropped images of all gels, and raw data for the chromatogram in panel d are available as source data.

Source data