Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates ∼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.
SignificanceRibosomes are found in every living organism, where they are responsible for the translation of messenger RNA into protein. The ribosome’s centrality to cell function is underscored by its evolutionary conservation; the core structure has changed little since its inception ∼4 billion years ago when ecosystems were anoxic and metal-rich. The ribosome is a model system for the study of bioinorganic chemistry, owing to the many highly coordinated divalent metal cations that are essential to its function. We studied the structure, function, and cation content of the ribosome under early Earth conditions (low O2, high Fe2+, and high Mn2+). Our results expand the roles of Fe2+ and Mn2+ in ancient and extant biochemistry as cofactors for ribosomal structure and function.
Preparing conventional DNA templates for in vitro RNA transcription involves PCR amplification of the DNA gene coding for the RNA of interest from plasmid or genomic DNA, subsequent amplification with primers containing a 5' T7 promoter region, and confirmation of the amplified DNA sequence. Complications arise in applications where long, nonnative sequences are desired in the final RNA transcript. Here we describe a ligase-independent method for the preparation of long synthetic DNA templates for in vitro RNA transcription. In Recursive PCR, partially complementary DNA oligonucleotides coding for the RNA sequence of interest are annealed, extended into the full-length double-stranded DNA, and amplified in a single PCR. Long insertions, mutations, or deletions are accommodated prior to in vitro transcription by simple substitution of oligonucleotides.
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
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