The Process of Infection with Bacteriophage øX174 XIII. Evidence for an Essential Bacterial "Site"

The burst of a starved bacterium infected with several øX174 bacteriophage was usually found to contain genetic traits of only one of the possible parents; less often, two phage multiplied in the same host cell. Unstarved cells, in contrast, supported the growth of at least four parental phage types. The unproductive phage seemed to be able to undergo the intracellular transition from parental single-stranded deoxyribonucleic acid to the double-stranded "replicative form" (RF). These results are taken to mean that some bacterial factor required for a step between RF synthesis and maturation of progeny is limited in starved cells

Familiar biological, chemical and physical events credibly evolve the Standard Genetic Code

The genetic code is profoundly shaped by an origin in ancient RNA-mediated interactions, needing an extended development to reach the Standard Genetic Code (SGC). That development can serially use RNA specificities, a ribonucleopeptide transition (RNPT), finally code escape and diaspora. An index of evolutionary plausibility based on least selection takes simultaneous account of speed and accuracy of evolution, identifying favored evolutions. Combining RNA world specificities allowed convergence of early coding to SGC assignments. Secondly, this was sufficient to launch a post-RNA-world RNPT. The RNPT allowed biosynthesis of complex amino acids, depending heavily on late code fusions between coexisting independent codes. Thirdly, escape from fluctuating, but highly-evolved codes of the RNPT applied a near-ideal selection for fastest-evolving and most accurate/useful genetic codes. Concurrently, a code and its microbial carrier suited to a free-living existence necessarily evolved. The established unity of life on Earth likely traces to SGC ascendancy during escape from the RNPT, and code diaspora.Comment: 18 partial pages of main text, 8 figures, some with panel

A diminutive and specific RNA binding site for L-tryptophan

Selection for amino acid affinity by elution of RNAs from tryptophan–Sepharose using free L-tryptophan evokes one sequence predominantly (K(D) = 12 µM), a symmetrical internal loop of 3 nt per side. Though we have also isolated larger sequences with affinity for tryptophan, successively squeezed selection in randomized tracts of 70, 60, 40, 20 and 17 nt show that this internal loop is the simplest sequence that can meet the column affinity selection. From sequence variation in ∼50 independent isolates, only 26 bits of information are required to describe this loop (equivalent to only 13 fully conserved nucleotides). Thus, it is among the simplest amino acid binding sites known, as well as selective among hydrophobic side chains. Among site sequences defined as essential to affinity by conservation, protection and modification-interference, there is a recurring CCA sequence (a tryptophan anticodon triplet) which apparently forms one side of the binding site. Such conserved juxtaposition of tryptophan with a cognate coding triplet supports a stereochemical origin for the genetic code

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

Specific RNA binding to ordered phospholipid bilayers

We have studied RNA binding to vesicles bounded by ordered and disordered phospholipid membranes. A positive correlation exists between bilayer order and RNA affinity. In particular, structure-dependent RNA binding appears for rafted (liquid-ordered) domains in sphingomyelin-cholesterol-1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. Binding to more highly ordered gel phase membranes is stronger, but much less RNA structure-dependent. All modes of RNA-membrane association seem to be electrostatic and headgroup directed. Fluorometry on 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes indicates that bound RNA broadens the gel-fluid melting transition, and reduces lipid headgroup order, as detected via fluorometric measurement of intramembrane electric fields. RNA preference for rafted lipid was visualized and confirmed using multiple fluorophores that allow fluorescence and fluorescence resonance energy transfer microscopy on RNA molecules closely associated with ordered lipid patches within giant vesicles. Accordingly, both RNA structure and membrane order could modulate biological RNA–membrane interactions

Abundance of correctly folded RNA motifs in sequence space, calculated on computational grids

Although functional RNA molecules are known to be biased in overall composition, the effects of background composition on the probability of finding a particular active site by chance has received little attention. The probability of finding a particular motif has important implications both for understanding the distribution of functional RNAs in ancient and modern organisms with varying genome compositions and for tuning SELEX pools to optimize the chance of finding specific functions. Here we develop a new method for calculating the probability of finding a modular motif containing base-paired regions, and use a computational grid to fold several hundred million random RNA sequences containing the core elements of the isoleucine aptamer and the hammerhead ribozyme to estimate the probability that a sequence containing these structural elements will fold correctly when isolated from background sequences of different compositions. We find that the two motifs are most likely to be found in distinct regions of compositional space, and that the regions of greatest abundance are influenced by the probability of finding the conserved bases, finding the flanking helices, and folding, in that order of importance. Additionally, we can refine our estimates of the number of random sequences required for a 50% probability of finding an example of each site in unbiased random pools of length 100 to 4.1 × 10(9) for the isoleucine aptamer and 1.6 × 10(10) for the hammerhead ribozyme. These figures are consistent with the facile recovery of these motifs from SELEX experiments

Evolution of the Standard Genetic Code

AbstractA 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.</jats:p

Packing the Standard Genetic Code in its box: 3-dimensional late Crick wobble

AbstractMinimally-evolved codes are constructed with randomly chosen Standard Genetic Code (SGC) triplets, and completed with completely random triplet assignments. Such “genetic codes” have not evolved, but retain SGC qualities. Retained qualities are inescapable, part of the logic of code evolution. For example, sensitivity of coding to arbitrary assignments, which must be &lt;≈ 10%, is intrinsic. Such sensitivity comes from elementary combinatorial properties of coding, and constrains any SGC evolution hypothesis. Similarly, evolution of last-evolved functions is difficult, due to late kinetic phenomena, likely common across codes. Census of minimally-evolved code assignments shows that shape and size of wobble domains controls packing 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 packing a highly ordered, degenerate code into a fixed three-dimensional space. Late Crick wobble in a 3-dimensional genetic code assembled by lateral transfer satisfies these varied, simultaneous requirements. By allowing parallel evolution of SGC domains, it 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 well-studied sources for order in amino acid coding, including a stereochemical minority of triplet-amino acid associations. Finally, fusion of its intermediates into the definitive SGC is credible, mirroring broadly-accepted later cellular evolution.</jats:p