Multiple prebiotic metals mediate translation

Abstract

15In extant biochemistry, Mg 2+ ions are essential for both structure and function of the ribosome (4,5) and for 16functions of many enzymes involved in translation (6). The translation system, which synthesizes all coded 17 protein (7), originated and matured during the Archean Eon (4-2.5 Ga; (8)). The common core of the 18 ribosome and many other aspects of the translation system have remained essentially frozen since the last 19 universal common ancestor (9,10). 20In ribosomes, structural Mg 2+ ions occur in rRNA-Mg 2+ clamps (11) (Fig. 1a), in dinuclear Mg 2+ -21 microclusters that frame the peptidyl transferase center (12) (Fig. 1b), and at the SSU-LSU interface (13) 22( Fig. 1c). Functional Mg 2+ ions stabilize a critical bend in mRNA between the P-site and A-site codons (14) 23 (Fig. 1d), and mediate rRNA-tRNA/mRNA interactions (15) (Fig. 1e, f 26All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 97RO). Thus, a total of 2.5 mM "background" Mg 2+ was present in each reaction (Fig. S1) MgCl2 and FeCl2 which were tested over a range of final concentrations (1, 3, 4, 5, 6, 7, 8, 9, and 11 mM; 122 All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. the amounts of metals in our assay reaction, which had no effect on DHFR activity (data not shown). 136Ribosome iron content. In order to measure the iron content of ribosomes, a 300 µL reaction was prepared 137 containing 45 µL of a 13.3 µM stock of E. coli ribosomes (New England Biolabs catalog # P0763S), 7 mM

Cutting in-line with iron: ribosomal function and non-oxidative RNA cleavage

“…Based on these findings, we proposed that early ribosomal folding and catalysis used Fe 2+ instead of, or in combination with Mg 2+ and other M 2+ ions. However, our observation of lower translation rates with anoxic Fe 2+ than with Mg 2+ (23) suggests that Fe 2+ might mediate non-oxidative damage of RNA at faster rates than Mg 2+ .…”

“…3) . Ribosome-associated Fe 2+ was quantified with TXRF, as described previously (23). Three non-pre-GOE growth conditions yielded ribosomes containing near background levels of Fe 2+ (< 2 mol Fe mol -1 ribosome).…”

“…Purified ribosomes were analyzed for iron content by total reflection X-ray fluorescence spectroscopy (TRXF) as described in Bray and Lenz et al (23).…”

“…Translation reactions were based on the methods of Bray and Lenz et al (23) with minor modifications. All 15 µL reactions contained 2.25 µL of purified ribosome samples normalized to 9 µg µL -1 (so that the final concentration of ribosomes in our reactions was 1.35 µg µL -1 ), 0.1 mM amino acid mix, 0.2 mM tRNAs, ∼0.2 µg µL -1 of dihydrofolate reductase mRNA, and 3 µL of factor mix (with RNA polymerase, and transcription/translation factors in 10 mM Mg 2+ ) from the PURExpress® Δ Ribosome Kit (New England Biolabs).…”

“…Under pre-GOE conditions, Fe 2+ would not have caused the oxidative damage to biomolecules that occurs today in the presence of O 2 , via Fenton chemistry (22). We recently reported that Fe 2+ can mediate in vitro translation under “pre-GOE” conditions: in the presence of abundant Fe 2+ and in the absence of O 2 (23,24). Based on these findings, we proposed that early ribosomal folding and catalysis used Fe 2+ instead of, or in combination with Mg 2+ and other M 2+ ions.…”