A crescendo of competent coding (c3) contains the Standard Genetic Code.

The Standard Genetic Code (SGC) can arise by fusion of partial codes evolved in different individuals, perhaps for differing prior tasks. Such code fragments can be unified into an SGC after later evolution of accurate third-position Crick wobble. Late wobble advent fills in the coding table, leaving only later development of translational initiation and termination to reach the SGC in separated domains of life. This code fusion mechanism is computationally implemented here. Late Crick wobble after C3 fusion (c3-lCw) is tested for its ability to evolve the SGC. Compared with previously studied isolated coding tables, or with increasing numbers of parallel, but nonfusing codes, c3-lCw reaches the SGC sooner, is successful in a smaller population, and presents accurate and complete codes more frequently. Notably, a long crescendo of SGC-like codes is exposed for selection of superior translation. c3-lCw also effectively suppresses varied disordered assignments, thus converging on a unified code. Such merged codes closely approach the SGC, making its selection plausible. For example: Under routine conditions, ≈1 of 22 c3-lCw environments evolves codes with ≥20 assignments and ≤3 differences from the SGC, notably including codes identical to the Standard Genetic Code.

Ordering events in a developing genetic code.

Preexisting partial genetic codes can fuse to evolve towards the complete Standard Genetic Code (SGC). Such code fusion provides a path of 'least selection', readily generating precursor codes that resemble the SGC. Consequently, such least selections produce the SGC via minimal, thus rapid, change. Optimal code evolution therefore requires delayed wobble. Early wobble encoding slows code evolution, very specifically diminishing the most likely SGC precursors: near-complete, accurate codes which are the products of code fusions. In contrast: given delayed wobble, the SGC can emerge from a truncation selection/evolutionary radiation based on proficient fused coding.

Fitting the standard genetic code into its triplet table.

Minimally evolved codes are constructed here; these have randomly chosen standard genetic code (SGC) triplets, completed with completely random triplet assignments. Such "genetic codes" have not evolved, but retain SGC qualities. Retained qualities are basic, part of the underpinning of coding. For example, the sensitivity of coding to arbitrary assignments, which must be < ∼10%, is intrinsic. Such sensitivity comes from the elementary combinatorial properties of coding and constrains any SGC evolution hypothesis. Similarly, assignment of last-evolved functions is difficult because of late kinetic phenomena, likely common across codes. Census of minimally evolved code assignments shows that shape and size of wobble domains controls the code's fit into a coding table, strongly shifting accuracy of codon assignments. Access to the SGC therefore requires a plausible pathway to limited randomness, avoiding difficult completion while fitting a highly ordered, degenerate code into a preset three-dimensional space. Three-dimensional late Crick wobble in a genetic code assembled by lateral transfer between early partial codes satisfies these varied, simultaneous requirements. By allowing parallel evolution of SGC domains, this origin can yield shortened evolution to SGC-level order and allow the code to arise in smaller populations. It effectively yields full codes. Less obviously, it unifies previously studied chemical, biochemical, and wobble order in amino acid assignment, including a stereochemical minority of triplet-amino acid associations. Finally, fusion of intermediates into the final SGC is credible, mirroring broadly accepted later cellular evolution.

Optimal Evolution of the Standard Genetic Code.

The Standard Genetic Code (SGC) exists in every known organism on Earth. SGC evolution via early unique codon assignment, then later wobble, yields coding resembling the near-universal code. Below, later wobble is shown to also create an optimal route to accurate codon assignment. Time of optimal codon assignment matches the previously defined mean time for ordered coding, exhibiting ≥ 90% of SGC order. Accurate evolution is also accessible, sufficiently frequent to appear in populations of 103 to 104 codes. SGC-like coding capacity, code order, and accurate assignments therefore arise together, in one attainable evolutionary intermediate. Examples, which plausibly resemble coding at evolutionary domain separation, are characterized.

Evolution of the Standard Genetic Code.

A near-universal Standard Genetic Code (SGC) implies a single origin for present Earth life. To study this unique event, I compute paths to the SGC, comparing different plausible histories. Notably, SGC-like coding emerges from traditional evolutionary mechanisms, and a superior route can be identified. To objectively measure evolution, progress values from 0 (random coding) to 1 (SGC-like) are defined: these measure fractions of random-code-to-SGC distance. Progress types are spacing/distance/delta Polar Requirement, detecting space between identical assignments/mutational distance to the SGC/chemical order, respectively. The coding system is based on selected RNAs performing aminoacyl-RNA synthetase reactions. Acceptor RNAs exhibit SGC-like Crick wobble; alternatively, non-wobbling triplets uniquely encode 20 amino acids/start/stop. Triplets acquire 22 functions by stereochemistry, selection, coevolution, or at random. Assignments also propagate to an assigned triplet's neighborhood via single mutations, but can also decay. A vast code universe makes futile evolutionary paths plentiful. Thus, SGC evolution is critically sensitive to disorder from random assignments. Evolution also inevitably slows near coding completion. The SGC likely avoided these difficulties, and two suitable paths are compared. In late wobble, a majority of non-wobble assignments are made before wobble is adopted. In continuous wobble, a uniquely advantageous early intermediate yields an ordered SGC. Revised coding evolution (limited randomness, late wobble, concentration on amino acid encoding, chemically conservative coevolution with a chemically ordered elite) produces varied full codes with excellent joint progress values. A population of only 600 independent coding tables includes SGC-like members; a Bayesian path toward more accurate SGC evolution is available.

Crick Wobble and Superwobble in Standard Genetic Code Evolution.

Wobble coding is inevitable during evolution of the Standard Genetic Code (SGC). It ultimately splits half of NN U/C/A/G coding boxes with different assignments. Further, it contributes to pervasive SGC order by reinforcing close spacing for identical SGC assignments. But wobble cannot appear too soon, or it will inhibit encoding and more decisively, obstruct evolution of full coding tables. However, these prior results assumed Crick wobble, NN U/C and NN A/G, read by a single adaptor RNA. Superwobble translates NN U/C/A/G codons, using one adaptor RNA with an unmodified 5' anticodon U (appropriate to earliest coding) in modern mitochondria, plastids, and mycoplasma. Assuming the SGC was selected when evolving codes most resembled it, characteristics of the critical selection events can be calculated. For example, continuous superwobble infrequently evolves SGC-like coding tables. So, continuous superwobble is a very improbable origin hypothesis. In contrast, late-arising superwobble shares late Crick wobble's frequent resemblance to SGC order. Thus late superwobble is possible, but yields SGC-like assignments less frequently than late Crick wobble. Ancient coding ambiguity, most simply, arose from Crick wobble alone. This is consistent with SGC assignments to NAN codons.

Eighty routes to a ribonucleotide world; dispersion and stringency in the decisive selection.

We examine the initial emergence of genetics; that is, of an inherited chemical capability. The crucial actors are ribonucleotides, occasionally meeting in a prebiotic landscape. Previous work identified six influential variables during such random ribonucleotide pooling. Geochemical pools can be in periodic danger (e.g., from tides) or constant danger (e.g., from unfavorable weather). Such pools receive Gaussian nucleotide amounts sporadically, at random times, or get varying substrates simultaneously. Pools use cross-templated RNA synthesis (5'-5' product from 5'-3' template) or para-templated (5'-5' product from 5'-5' template) synthesis. Pools can undergo mild or strong selection, and be recently initiated (early) or late in age. Considering >80 combinations of these variables, selection calculations identify a superior route. Most likely, an early, sporadically fed, cross-templating pool in constant danger, receiving ≥1 mM nucleotides while under strong selection for a coenzyme-like product, will host selection of the first encoded biochemical functions. Predominantly templated products emerge from a critical event, the starting bloc selection, which exploits inevitable differences among early pools. Favorable selection has a simple rationale; it is increased by product dispersion (SD/mean), by selection intensity (mild or strong), or by combining these factors as stringency, reciprocal fraction of pools selected (1/sfsel). To summarize: chance utility, acting via a preference for disperse, templated coenzyme-like dinucleotides, uses stringent starting bloc selection to quickly establish majority encoded/genetic expression. Despite its computational origin, starting bloc selection is largely independent of specialized assumptions. This ribodinucleotide route to inheritance may also have facilitated 5'-3' chemical RNA replication.

Non-Watson-Crick RNA synthesis suited to origin functions.

A templated RNA synthesis is characterized in which G5'pp5'G accelerates synthesis of A5'pp5'A from pA and chemically activated ImpA precursors. Similar acceleration is not observable in the presence of UppU, CppC, AppG, AppA, or pG alone. Thus, it seems likely that AppA is templated by GppG via a form or forms of G:A base-pairing. AppA also appears, more slowly, via a previously known untemplated second-order chemical route. Such AppA synthesis requires only ordinary near-neutral solutions containing monovalent and divalent salts, and rates are only slightly sensitive to variation in pH. Templated synthesis rates are first order in pA, ImpA, and template GppG; thus third order overall. Therefore, this reaction resembles cross-templating of AppA on poly(U), but is notably slower and less sensitive to temperature. Viewing AppA as a coenzyme analog, GppG templating provides a simpler molecular route, termed para-templating, to encoded chemical functions. Para-templating can also arise from a single, localized nucleobase geosynthetic event which yields purines. It requires only a single backbone-forming chemistry. Thus it may have appeared earlier and served as evolutionary precursor for more complex forms of encoded genetic expression.

Cross-backbone templating; ribodinucleotides made on poly(C).

G(5')pp(5')G synthesis from pG and chemically activated 2MeImpG is accelerated by the addition of complementary poly(C), but affected only slightly by poly(G) and not at all by poly(U) and poly(A). This suggests that 3'-5' poly(C) is a template for uncatalyzed synthesis of 5'-5' GppG, as was poly(U) for AppA synthesis, previously. The reaction occurs at 50 mM mono- and divalent ion concentrations, at moderate temperatures, and near pH 7. The reactive complex at the site of enhanced synthesis of 5'-5' GppG seems to contain a single pG, a single phosphate-activated nucleotide 2 MeImpG, and a single strand of poly(C). Most likely this structure is base-paired, as the poly(C)-enhanced reaction is completely disrupted between 30 and 37 °C, whereas slower, untemplated synthesis of GppG accelerates. More specifically, the reactive center acts as would be expected for short, isolated G nucleotide stacks expanded and ordered by added poly(C). For example, poly(C)-mediated GppG production is very nonlinear in overall nucleotide concentration. Uncatalyzed NppN synthesis is now known for two polymers and their complementary free nucleotides. These data suggest that varied, simple, primordial 3'-5' RNA sequences could express a specific chemical phenotype by encoding synthesis of complementary, reactive, coenzyme-like 5'-5' ribodinucleotides.

Efficient Heritable Gene Expression Readily Evolves in RNA Pools.

Heritable gene expression arises readily in a simple non-genetic system employing known small-RNA biochemistry. Pooled cross-templating ribonucleotides show varied chemical competence on which selection acts, even calculating only minimal effects. Evolution can be quick-computed progress toward encoded gene expression can require only days or weeks for two millimolar, partly activated complementary 5' ribonucleotides. After only one product selection cycle, early templating can become prevailing pool behavior. Subsequently, a selected templated product is efficiently amplified as a pool ages, frequently accumulated in the same order of concentration as incoming nucleotides. Pools spontaneously favor templating because sporadic nucleotide accumulations increase it-and selection increases templating in pools of all ages. Nonetheless, templated chemical competence appears most easily in young pools. Pool history is critical-pools can perish from periodic hazards (like tides), or alternatively, from hazards roughly constant in time (like rainfall). Selection is greatly enhanced in constant hazard pools-more effective if pools have varied ages. Stronger selection is disproportionately more effective. Selected evolutionary change has an uncomplicated molecular basis-progress from chemical product synthesis to templated, proto-genetic inheritance exploits identity between templating and entropic catalysis. Though discovered by computation, selection of an elevated product of template catalysis is plausible, independent of any chemical or mathematical assumption. Selected chemical variation before genetics (chance utility) therefore inaugurates inheritance, even when hindered by unstable, dilute nucleotides, erratically supplied in undependable quantities. Remarkably, such uncontrolled conditions are not necessarily hostile, but can instead accelerate appearance of primordial gene-like behavior.

Poly(U) RNA-templated synthesis of AppA.

Simple nucleotide templating activities are of interest as potential primordial reactions. Here we describe the acceleration of 5'-5' AppA synthesis by 3'-5' poly(U) under normal solution conditions. This reaction is apparently templated via complementary U:A base-pairing, despite the involvement of two different RNA backbones, because poly(U), unlike other polymers, significantly stimulates AppA synthesis. These interactions occur in moderate (K(+)) and (Mg(2+)) and are temperature sensitive, being more efficient at 10°C than at 4°C, but absent at 20°C. The reaction is only slightly pH sensitive, despite potentially relevant substrate pKa's. Kinetic data explicitly support production of AppA by interaction of stacked 2MeImpA and pA nucleotides paired with a single molecule of U template. At a lower rate, AppA can also be produced by a chemical reaction between 2MeImpA and pA, without participation of poly(U). Molecular modeling suggests that 5'-5' joining between stacked or concurrently paired A's can occur without major departures from normal U-A helical coordinates. So, coenzyme-like 5'-5' purine dinucleotides might be readily synthesized from 3'-5' RNAs with complementary sequences.

Biochemical Refinement Before Genetics: Chance Utility.

Given two primordial conditions that seem likely to be common, near-ideal reactions for evolutionary progress are realized. These requisites are sporadic availability of pooled reactants and evolutionarily useful products within a pool's repertoire. These intrinsically optimizing circumstances function without genetics, and therefore can help evolve a first genetic system. This process is termed chance utility.

Human tRNA(Sec) associates with HeLa membranes, cell lipid liposomes, and synthetic lipid bilayers.

We have shown previously that simple RNA structures bind pure phospholipid liposomes. However, binding of bona fide cellular RNAs under physiological ionic conditions is shown here for the first time. Human tRNA(Sec) contains a hydrophobic anticodon-loop modification: N6-isopentenyladenosine (i6A) adjacent to its anticodon. Using a highly specific double-probe hybridization assay, we show mature human tRNA(Sec) specifically retained in HeLa intermediate-density membranes. Further, isolated human tRNA(Sec) rebinds to liposomes from isolated HeLa membrane lipids, to a much greater extent than an unmodified tRNA(Sec) transcript. To better define this affinity, experiments with pure lipids show that liposomes forming rafts or including positively charged sphingosine, or particularly both together, exhibit increased tRNA(Sec) binding. Thus tRNA(Sec) residence on membranes is determined by several factors, such as hydrophobic modification (likely isopentenylation of tRNA(Sec)), lipid structure (particularly lipid rafts), or sphingosine at a physiological concentration in rafted membranes. From prior work, RNA structure and ionic conditions also appear important. tRNA(Sec) dissociation from HeLa liposomes implies a mean membrane residence of 7.6 min at 24°C (t(1/2) = 5.3 min). Clearly RNA with a 5-carbon hydrophobic modification binds HeLa membranes, probably favoring raft domains containing specific lipids, for times sufficient to alter biological fates.

A ribonucleotide Origin for Life--fluctuation and near-ideal reactions.

Oligoribonucleotides are potentially capable of Darwinian evolution - they may replicate and can express an independent chemical phenotype, as embodied in modern enzymatic cofactors. Using quantitative chemical kinetics on a sporadically fed ribonucleotide pool, unreliable supplies of unstable activated ribonucleotides A and B at low concentrations recurrently yield a replicating AB polymer with a potential chemical phenotype. Self-complementary replication in the pool occurs during a minority (here ≈ 35 %) of synthetic episodes that exploit coincidental overlaps between 4, 5 or 6 spikes of arbitrarily arriving substrates. Such uniquely productive synthetic episodes, in which near-ideal reaction sequences recur at random, account for most AB oligonucleotide synthesis, and therefore underlie the emergence of net replication under realistic primordial conditions. Because overlapping substrate spikes are unexpectedly frequent, and in addition, complex spike sequences appear disproportionately, a sporadically fed pool can host unexpectedly complex syntheses. Thus, primordial substrate fluctuations are not necessarily a barrier to Darwinism, but instead can facilitate early evolution.

Multiple translational products from a five-nucleotide ribozyme.

An indispensable step in protein biosynthesis is the 2(')(3(')) aminoacylation of tRNA by aminoacyl-tRNA synthetases. Here we show that a similar activity exists in a tiny, 5-nt-long RNA enzyme with a 3-nt active center. The small ribozyme initially trans-phenylalanylates a partially complementary 4-nt RNA selectively at its terminal 2(')-ribose hydroxyl using PheAMP, the natural form for activated amino acid. The initial 2(') Phe-RNA product can be elaborated into multiple peptidyl-RNAs. Reactions do not require divalent cations, and have limited dependence on monovalent cations. Small size and minimal requirements for regiospecific translational activity strongly support the hypothesis that minuscule RNA enzymes participated in early forms of translation.

The Genetic Code Assembles via Division and Fusion, Basic Cellular Events.

Standard Genetic Code (SGC) evolution is quantitatively modeled in up to 2000 independent coding 'environments'. Environments host multiple codes that may fuse or divide, with division yielding identical descendants. Code division may be selected-sophisticated gene products could be required for an orderly separation that preserves the coding. Several unforeseen results emerge: more rapid evolution requires unselective code division rather than its selective form. Combining selective and unselective code division, with/without code fusion, with/without independent environmental coding tables, and with/without wobble defines 25 = 32 possible pathways for SGC evolution. These 32 possible histories are compared, specifically, for evolutionary speed and code accuracy. Pathways differ greatly, for example, by ≈300-fold in time to evolve SGC-like codes. Eight of thirty-two pathways employing code division evolve quickly. Four of these eight that combine fusion and division also unite speed and accuracy. The two most precise, swiftest paths; thus the most likely routes to the SGC are similar, differing only in fusion with independent environmental codes. Code division instead of fusion with unrelated codes implies that exterior codes can be dispensable. Instead, a single ancestral code that divides and fuses can initiate fully encoded peptide biosynthesis. Division and fusion create a 'crescendo of competent coding', facilitating the search for the SGC and also assisting the advent of otherwise uniformly disfavored wobble coding. Code fusion can unite multiple codon assignment mechanisms. However, via code division and fusion, an SGC can emerge from a single primary origin via familiar cellular events.

The plausibility of RNA-templated peptides: simultaneous RNA affinity for adjacent peptide side chains.

According to the RNA world hypothesis, coded peptide synthesis (translation) must have been first catalyzed by RNAs. Here, we show that small RNA sequences can simultaneously bind the dissimilar amino acids His and Phe in peptide linkage. We used in vitro counterselection/selection to isolate a pool of RNAs that bind the dipeptide NH(2)-His-Phe-COOH with K (D) ranging from 36 to 480 µM. These sites contact both side chains, usually including the protonated imidazole of His, but bind-free L: -His and L: -Phe with much lower, sometimes undetectable, affinities. The most frequent His-Phe sites do not usually contain previously isolated sites for individual amino acids, and are only ≈35 % larger than previously known separate His and Phe sites. Nonetheless, His-Phe sites appear enriched in His anticodons, as previous L: -His sites also were. Accordingly, these data add to existing experimental evidence for a stereochemical genetic code. In these peptide sites, bound amino acids approach each other to a proximity that allows a covalent peptide linkage. Isolation of several RNAs embracing two amino acids with a linking peptide bond supports the idea that a direct-RNA-template could encode primordial peptides, though crucial experiments remain.

Simple, recurring RNA binding sites for L-arginine.

Seven new arginine binding motifs have been selected from a heterogeneous RNA pool containing 17, 25, and 50mer randomized tracts, yielding 131 independently derived binding sites that are multiply isolated. The shortest 17mer random region is sufficient to build varied arginine binding sites using five different conserved motifs (motifs 1a, 1b, 1c, 2, and 4). Dissociation constants are in the fractional millimolar to millimolar range. Binding sites are amino acid side-chain specific and discriminate moderately between L- and D-stereoisomers of arginine, suggesting a molecular focus on side-chain guanidinium. An arginine coding triplet (codon/anticodon) is highly conserved within the largest family of Arg sites (72% of all sequences), as has also been found in minimal, most prevalent RNA binding sites for Ile, His, and Trp.

Chiral histidine selection by D-ribose RNA.

The invariant choice of L-amino acids and D-ribose RNA for biological translation requires explanation. Here we study this chiral choice using mixed, equimolar D-ribose RNAs having 15, 18, 21, 27, 35, and 45 contiguous randomized nucleotides. These are used for simultaneous affinity selection of the smallest bound and eluted RNAs using equal amounts of L- and D-His immobilized on an achiral glass support, with racemic histidine elution. The experiment as a whole therefore determines whether RNA containing D-ribose binds L-histidine or D-histidine more easily (that is, by using a site that is more abundant/requires fewer nucleotides). The most prevalent/smallest RNA sites are reproducibly and repeatedly selected and there is a four- to sixfold greater abundance of L-histidine sites. RNA's chiral D-ribose therefore yields a more frequent fit to L-histidine. Accordingly, a D-ribose RNA site for L-His is smaller by the equivalent of just over one conserved nucleotide. The most prevalent L-His site also performs better than the most frequent D-His site-but rarer D-ribose RNAs can bind D-His with excellent affinity and discrimination. The prevalent L-His site is one we have selected before under very different conditions. Thus, selection is again reproducible, as is the recurrence of cognate coding triplets in these most probable L-His sites. If our selected RNA population were equilibrated with racemic His, we calculate that L-His would participate in seven of eight His:RNA complexes, or more. Thus, if D-ribose RNA were first chosen biologically, translational L-His usage could have followed.