A general framework for conventional models of the origin of life (OOL) is the specification of a ‘privileged function.’ A privileged function is an extant biological function that is excised from its biological context, elevated in importance over other functions, and transported back in time to a primitive chemical or geological environment. In RNA or Clay Worlds, the privileged function is replication. In Metabolism-First Worlds, the privileged function is metabolism. In Thermal Vent Worlds, the privileged function is energy harvesting from chemical gradients. In Membrane Worlds, the privileged function is compartmentalization. In evaluating these models, we consider the contents and properties of the Universal Gene Set of life, which is the set of orthologous genes conserved throughout the tree of life and found in every living system. We also consider the components and properties of the Molecular Toolbox of Life, which contains twenty amino acids, eight nucleotides, glucose, polypeptide, polynucleotide, and several other components. OOL models based on privileged functions necessarily depend on “takeovers” to transition from previous genetic and catalytic systems to the extant DNA/RNA/protein system, requiring replacement of one Molecular Toolbox with another and of one Universal Gene Set with another. The observed robustness and contents of the Toolbox of Life and the Universal Gene Set over the last 3.7 billion years are thought to be post hoc phenomena. Once the takeover processes are acknowledged and are reasonably considered, the privileged function models are seen to be extremely complex with low predictive power. These models require indeterminacy and plasticity of biological and chemical processes.

The assembled bacterial ribosome contains around 50 proteins and many counterions. Here, focusing on rRNA from the large ribosomal subunit, we demonstrate that Mg2+ causes structural collapse in the absence of ribosomal proteins. The collapsed rRNA forms many native-like RNA–RNA interactions, similar to those observed in the assembled ribosome. We assayed rRNA structure by chemical footprinting in the presence and absence of Mg2+. Our results indicate that Mg2+-dependent conformational change is focused in non-helical regions, consistent with tertiary interactions. In the presence of Mg2+, the large subunit rRNA adopts a state that includes the core inter-domain architecture of the assembled ribosome. We infer that the rRNA–Mg2+ state represents the core architecture of the LSU which, while not catalytically active, positions the residues of the LSU rRNA in such a way as to promote native interactions with rProteins to ultimately form a functional LSU.

We have proposed that the ancient ribosome increased in size during early evolution by addition of small folding-competent RNAs. In this Accretion Model, small RNAs and peptides were subsumed onto subunit surfaces, gradually encasing and freezing previously acquired components. The model predicts that appropriate rRNA fragments have inherited local autonomy of folding and local autonomy of assembly with ribosomal proteins (rProteins), and that the rProtein and rRNA are co-chaperones. To test these predictions, we investigate the rRNA interactions of rProtein uL23 and its tail, uL23tail, which is a β-hairpin that penetrates deep into the core of the large ribosomal subunit. In the assembled ribosome, uL23tail associates with Domain III of the rRNA and a subdomain called “DIIIcore”. Here using band shift assays, fluorescence Job plots, and yeast three-hybrid assays, we investigate the interactions of rProtein uL23 and its tail with Domain III and with DIIIcore rRNA. We observe rRNA1-uL23tail1 complexes in the absence of Mg2+ ions and rRNA1-uL23tailn (n > 1) complexes in the presence of Mg2+ ions. By contrast, the intact uL23 rProtein binds in slightly anticooperative complexes of various stoichiometries. The globular and tail regions of rProtein uL23 are distinctive in their folding behaviors and the ion dependences of their association with rRNA. For the globular region of the rProtein, folding is independent of rRNA, and rRNA association is predominantly by nonelectrostatic mechanisms. For the tail region of the protein, folding requires rRNA, and association is predominantly by electrostatic mechanisms. We believe these protein capabilities could have roots in ancient evolution and could be mechanistically important in co-chaperoning the assembly of the ribosome.

As illustrated by the mitochondrion and the eukaryotic cell, little in biology makes sense except in light of mutualism. Mutualisms are persistent, intimate, and reciprocal exchanges; an organism proficient in obtaining certain benefits confers those on a partner, which reciprocates by conferring different benefits. Mutualisms (i) increase fitness, (ii) inspire robustness, (iii) are resilient and resistant to change, (iv) sponsor co-evolution, (v) foster innovation, and (vi) involve partners that are distantly related with contrasting yet complementary proficiencies. Previous to this work, mutualisms were understood to operate on levels of cells, organisms, ecosystems, and even societies and economies. Here, the concepts of mutualism are extended to molecules and are seen to apply to the relationship between RNA and protein. Polynucleotide and polypeptide are Molecules in Mutualism. RNA synthesizes protein in the ribosome and protein synthesizes RNA in polymerases. RNA and protein are codependent, and trade proficiencies. Protein has proficiency in folding into complex three-dimensional states, contributing enzymes, fibers, adhesives, pumps, pores, switches, and receptors. RNA has proficiency in direct molecular recognition, achieved by complementary base pairing interactions, which allow it to maintain, record, and transduce information. The large phylogenetic distance that characterizes partnerships in organismal mutualism has close analogy with large distance in chemical space between RNA and protein. The RNA backbone is anionic and self-repulsive and cannot form hydrophobic structural cores. The protein backbone is neutral and cohesive and commonly forms hydrophobic cores. Molecules in Mutualism extends beyond RNA and protein. A cell is a consortium of molecules in which nucleic acids, proteins, polysaccharides, phospholipids, and other molecules form a mutualism consortium that drives metabolism and replication. Analogies are found in systems such as stromatolites, which are large consortia of symbiotic organisms. It seems reasonable to suggest that ‘polymers in mutualism relationships’ is a useful and predictive definition of life.

In a model describing the origin and evolution of the translation system, ribosomal RNA (rRNA) grew in size by accretion [Petrov, A. S., et al. (2015) History of the Ribosome and the Origin of Translation. Proc. Natl. Acad. Sci. U.S.A. 112, 15396–15401]. Large rRNAs were built up by iterative incorporation and encasement of small folded RNAs, in analogy with addition of new LEGOs onto the surface of a preexisting LEGO assembly. In this model, rRNA robustness in folding arises from inherited autonomy of local folding. We propose that rRNAs can be decomposed at various granularities, retaining folding mechanism and folding competence. To test these predictions, we disassembled Domain III of the large ribosomal subunit (LSU). We determined whether local rRNA structure, stability, and folding pathways are autonomous. Thermal melting, chemical footprinting, and circular dichroism were used to infer rules that govern folding of rRNA. We deconstructed Domain III of the LSU rRNA by mapping out its complex multistep melting pathway. We studied Domain III and two equal-size “sub-Domains” of Domain III. The combined results are consistent with a model in which melting transitions of Domain III are conserved upon cleavage into sub-Domains. Each of the eight melting transitions of Domain III corresponds in Tm and ΔH with a transition observed in one of the two isolated sub-Domains. The results support a model in which structure, stability, and folding mechanisms are dominated by local interactions and are unaffected by separation of the sub-Domains. Domain III rRNA is distinct from RNAs that form long-range cooperative interaction networks at early stages of folding or that do not fold reversibly.

An RNA World that predated the modern world of polypeptide and polynucleotide is one of the most widely accepted models in origin of life research. In this model, the translation system shepherded the RNA World into the extant biology of DNA, RNA, and protein. Here, we examine the RNA World Hypothesis in the context of increasingly detailed information available about the origins, evolution, functions, and mechanisms of the translation system. We conclude that the translation system presents critical challenges to RNA World Hypotheses. Firstly, a timeline of the RNA World is problematic when the ribosome is incorporated. The mechanism of peptidyl transfer of the ribosome appears distinct from evolved enzymes, signaling origins in a chemical rather than biological milieu. Secondly, we have no evidence that the basic biochemical toolset of life is subject to substantive change by Darwinian evolution, as required for the transition from the RNA world to extant biology. Thirdly, we do not see specific evidence for biological takeover of ribozyme function by protein enzymes. Finally, we can find no basis for preservation of the ribosome as ribozyme or the universality of translation, if it were the case that other information transducing ribozymes, such as ribozyme polymerases, were replaced by protein analogs and erased from the phylogenetic record. We suggest that an updated model of the RNA World should address the current state of knowledge of the translation system.

We present a molecular-level model for the origin and evolution of the translation system, using a 3D comparative method. In this model, the ribosome evolved by accretion, recursively adding expansion segments, iteratively growing, subsuming, and freezing the rRNA. Functions of expansion segments in the ancestral ribosome are assigned by correspondence with their functions in the extant ribosome. The model explains the evolution of the large ribosomal subunit, the small ribosomal subunit, tRNA, and mRNA. Prokaryotic ribosomes evolved in six phases, sequentially acquiring capabilities for RNA folding, catalysis, subunit association, correlated evolution, decoding, energy-driven translocation, and surface proteinization. Two additional phases exclusive to eukaryotes led to tentacle-like rRNA expansions. In this model, ribosomal proteinization was a driving force for the broad adoption of proteins in other biological processes. The exit tunnel was clearly a central theme of all phases of ribosomal evolution and was continuously extended and rigidified. In the primitive noncoding ribosome, proto-mRNA and the small ribosomal subunit acted as cofactors, positioning the activated ends of tRNAs within the peptidyl transferase center. This association linked the evolution of the large and small ribosomal subunits, proto-mRNA, and tRNA.

The origins and evolution of the ribosome, 3–4 billion years ago, remain imprinted in the biochemistry of extant life and in the structure of the ribosome. Processes of ribosomal RNA (rRNA) expansion can be “observed” by comparing 3D rRNA structures of bacteria (small), yeast (medium), and metazoans (large). rRNA size correlates well with species complexity. Differences in ribosomes across species reveal that rRNA expansion segments have been added to rRNAs without perturbing the preexisting core. Here we show that rRNA growth occurs by a limited number of processes that include inserting a branch helix onto a preexisting trunk helix and elongation of a helix. rRNA expansions can leave distinctive atomic resolution fingerprints, which we call “insertion fingerprints.” Observation of insertion fingerprints in the ribosomal common core allows identification of probable ancestral expansion segments. Conceptually reversing these expansions allows extrapolation backward in time to generate models of primordial ribosomes. The approach presented here provides insight to the structure of pre-last universal common ancestor rRNAs and the subsequent expansions that shaped the peptidyl transferase center and the conserved core. We infer distinct phases of ribosomal evolution through which ribosomal particles evolve, acquiring coding and translocation, and extending and elaborating the exit tunnel.

Some of the magnesium ions in the ribosome are coordinated by multiple rRNA phosphate groups. These magnesium ions link distal sequences of rRNA, primarily by incorporating phosphate groups into the first coordination shell. Less frequently, magnesium interacts with ribosomal proteins. Ribosomal protein L2 appears to be unique by forming specific magnesium-mediated interactions with rRNA. Using optimized models derived from X-ray structures, we subjected rRNA/magnesium/water/rProtein L2 assemblies to quantum mechanical analysis using the density functional theory and natural energy decomposition analysis. The combined results provide estimates of energies of formation of these assemblies, and allow us to decompose the energies of interaction. The results indicated that RNA immobilizes magnesium by multidentate chelation with phosphate, and that the magnesium ions in turn localize and polarize water molecules, increasing energies and specificities of interaction of these water molecules with L2 protein. Thus, magnesium plays subtle, yet important, roles in ribosomal assembly beyond neutralization of electrostatic repulsion and direct coordination of RNA functional groups.

In RNA, A-form helices are commonly terminated by tetraloops or 3′ dangling ends. Aside from helices themselves, these helix-breaking motifs appear to be among the most frequent and repetitive structural elements of large folded RNAs. We show here that within a frequent type of tetraloop, cGNRAg (G is guanine, N is any base, R is purine, A is adenine), a tension exists between the backbone torsional energy of the loop and the energy contributed by molecular interactions (stacking and pairing). A model in which favorable bond rotamers are opposed by favorable stacking and pairing interactions is consistent with our observation that release of torsional restraints upon conversion of one or more loop riboses to more flexible trimethylene phosphate(s) contributes favorably to the enthalpy of folding. This effect presumably results from improved stacking and hydrogen-bonding interactions upon release of torsional restraints. The most obvious possibility for improving molecular interactions is a repositioning of A, which is proximal to the unfavorable torsion angles in native cGNRAg tetraloops, and which is unstacked on the 3′ side and unpaired (it forms a single hydrogen bond with the opposing G). This tension between favorable bond rotamers and favorable molecular interactions may be representative of a general evolutionary strategy to prevent achievement of deep and irreversible thermodynamic wells in folded RNAs. Finally, we observe a simple stacking substructure with conserved geometry and sequence that forms a scaffold for both tetraloops and 3′ dangling ends. It seems that simple substructures can build RNA motifs, which combine to establish the fundamental architecture of RNA.

The conversion of a nucleic acid from single strands to double strands is thought to involve slow nucleation followed by fast double-strand propagation. Here, for RNA double-strand propagation, we propose an atomic resolution reaction mechanism. This mechanism, called the stack-ratchet, is based on data-mining of three-dimensional structures and on available thermodynamic information. The stack-ratchet mechanism extends and adds detail to the classic zipper model proposed by Porschke (Porschke, D. Biophysical Chemistry 1974, 2, pp. 97−101). Porschke’s zipper model describes the addition of a base pair to a nucleated helix in terms of a single type of elementary reaction; a concerted process in which the two bases, one from each strand, participate in the transition state. In the stack-ratchet mechanism proposed here a net base-pairing step consists of two elementary reactions. Motions of only one strand are required to achieve a given transition state. One elementary reaction preorganizes and stacks the 3′ single-strand, driven by base−base stacking interactions. A second elementary reaction stacks the 5′ strand and pairs it with the preorganized 3′ strand. In the stack-ratchet mechanism, a variable length 3′ stack leads the single-strand/double-strand junction. The stack-ratchet mechanism is not a two-state process. A base can be (i) unstacked and unpaired, (ii) stacked and paired, or (ii) stacked and unpaired (only on the 3′ strand). The data suggests that helices of DNA and of RNA do not propagate by similar mechanisms.