Folding, Assembly, and Persistence: The Essential Nature and Origins of Biopolymers.

Life as we know it requires three basic types of polymers: polypeptide, polynucleotide, and polysaccharide. Here we evaluate both universal and idiosyncratic characteristics of these biopolymers. We incorporate this information into a model that explains much about their origins, selection, and early evolution. We observe that all three biopolymer types are pre-organized, conditionally self-complementary, chemically unstable in aqueous media yet persistent because of kinetic trapping, with chiral monomers and directional chains. All three biopolymers are synthesized by dehydration reactions that are catalyzed by molecular motors driven by hydrolysis of phosphorylated nucleosides. All three biopolymers can access specific states that protect against hydrolysis. These protected states are folded, using self-complementary interactions among recurrent folding elements within a given biopolymer, or assembled, in associations between the same or different biopolymer types. Self-association in a hydrolytic environment achieves self-preservation. Heterogeneous association achieves partner-preservation. These universal properties support a model in which life's polymers emerged simultaneously and co-evolved in a common hydrolytic milieu where molecular persistence depended on folding and assembly. We believe that an understanding of the structure, function, and origins of any given type of biopolymer requires the context of other biopolymers.

The Central Symbiosis of Molecular Biology: Molecules in Mutualism.

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.

Ancestral Interactions of Ribosomal RNA and Ribosomal Proteins.

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 ß-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.

Iron mediates catalysis of nucleic acid processing enzymes: support for Fe(II) as a cofactor before the great oxidation event.

Life originated in an anoxic, Fe2+-rich environment. We hypothesize that on early Earth, Fe2+ was a ubiquitous cofactor for nucleic acids, with roles in RNA folding and catalysis as well as in processing of nucleic acids by protein enzymes. In this model, Mg2+ replaced Fe2+ as the primary cofactor for nucleic acids in parallel with known metal substitutions of metalloproteins, driven by the Great Oxidation Event. To test predictions of this model, we assay the ability of nucleic acid processing enzymes, including a DNA polymerase, an RNA polymerase and a DNA ligase, to use Fe2+ in place of Mg2+ as a cofactor during catalysis. Results show that Fe2+ can indeed substitute for Mg2+ in catalytic function of these enzymes. Additionally, we use calculations to unravel differences in energetics, structures and reactivities of relevant Mg2+ and Fe2+ complexes. Computation explains why Fe2+ can be a more potent cofactor than Mg2+ in a variety of folding and catalytic functions. We propose that the rise of O2 on Earth drove a Fe2+ to Mg2+ substitution in proteins and nucleic acids, a hypothesis consistent with a general model in which some modern biochemical systems retain latent abilities to revert to primordial Fe2+-based states when exposed to pre-GOE conditions.

The Origin of Life: Models and Data.

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.

Frozen in Time: The History of Proteins.

The ribosome is imprinted with a detailed molecular chronology of the origins and early evolution of proteins. Here we show that when arranged by evolutionary phase of ribosomal evolution, ribosomal protein (rProtein) segments reveal an atomic level history of protein folding. The data support a model in which aboriginal oligomers evolved into globular proteins in a hierarchical step-wise process. Complexity of assembly and folding of polypeptide increased incrementally in concert with expansion of rRNA. (i) Short random coil proto-peptides bound to rRNA, and (ii) lengthened over time and coalesced into ß-ß secondary elements. These secondary elements (iii) accreted and collapsed, primarily into ß-domains. Domains (iv) accumulated and gained complex super-secondary structures composed of mixtures of α-helices and ß-strands. Early protein evolution was guided and accelerated by interactions with rRNA. rRNA and proto-peptide provided mutual protection from chemical degradation and disassembly. rRNA stabilized polypeptide assemblies, which evolved in a stepwise process into globular domains, bypassing the immense space of random unproductive sequences. Coded proteins originated as oligomers and polymers created by the ribosome, on the ribosome and for the ribosome. Synthesis of increasingly longer products was iteratively coupled with lengthening and maturation of the ribosomal exit tunnel. Protein catalysis appears to be a late byproduct of selection for sophisticated and finely controlled assembly.

Imprint of Ancient Evolution on rRNA Folding.

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.

History of the ribosome and the origin of translation.

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.