92207040
Topological Structures Influencing Kinetic Control in Small, Catalytically Closed, RNA Recombinase Systems Emerging from the Spontaneous Self-Organization of Heterogeneous Fragments of the Azoarcus Ribozyme
..........................................................................3 The Origins of Biological Systems...........................................4 Autocatalysis and Self-Replication..........................................9 Azoarcus Group I Ribozyme.................................................15 Experimental Methods........................................................20 3-Genotype Network Topologies............................................21 Results and Discussion........................................................27 Conclusion.......................................................................40 References........................................................................42
15234896
Highly efficient self-replicating RNA enzymes.
An RNA enzyme has been developed that catalyzes the joining of oligonucleotide substrates to form additional copies of itself, undergoing self-replication with exponential growth. The enzyme also can cross-replicate with a partner enzyme, resulting in their mutual exponential growth and enabling self-sustained Darwinian evolution. The opportunity for inventive evolution within this synthetic genetic system depends on the diversity of the evolving population, which is limited by the catalytic efficiency of the enzyme. Directed evolution was used to improve the efficiency of the enzyme and increase its exponential growth rate to 0.14 min(-1), corresponding to a doubling time of 5 min. This is close to the limit of 0.21 min(-1) imposed by the rate of product release, but sufficient to enable more than 80 logs of growth per day.
30031582
Self-assembly of a group I intron from inactive oligonucleotide fragments.
The Azoarcus group I ribozyme was broken into four fragments, 39-63 nucleotides long, that can self-assemble into covalently contiguous ribozymes via RNA-directed recombination events. The fragments have no activity individually yet can cooperate through base pairing and tertiary interactions to produce stable trans complexes at 48 degrees C. These complexes can then catalyze a sequence of energy-neutral recombination reactions utilizing other oligomers as substrates, assembling covalent versions of the ribozyme. Up to 17% of the original fragments are converted into approximately 200 nucleotide products in 8 hr. Assembly occurs primarily by only one of many possible pathways, and the reaction is driven in the correct and forward direction by the burial of key base-pairing regions in stems after recombination. Autocatalysis, and hence self-replication, is inferred by a reaction rate increase upon doping the reaction with full-length RNA.
42407804
Spontaneous Covalent Self‐Assembly of the Azoarcus Ribozyme from Five Fragments
Spontaneous covalent assembly of short RNA fragments has been proposed as a plausible prebiotically relevant pathway to a self‐reproducing system. We previously showed that the Azoarcus group I intron could self‐assemble from four RNA fragments. Here, we extended this fragmentation to five RNAs that averaged <40 nucleotides in length. We optimized this reaction and showed that a dehydration–rehydration sequence was the most effective means to date to shift the self‐assembly equilibrium from reactants to products.
4939632
Prebiotic chemistry and the origin of the RNA world.
The demonstration that ribosomal peptide synthesis is a ribozyme-catalyzed reaction makes it almost certain that there was once an RNA World. The central problem for origin-of-life studies, therefore, is to understand how a protein-free RNA World became established on the primitive Earth. We first review the literature on the prebiotic synthesis of the nucleotides, the nonenzymatic synthesis and copying of polynucleotides, and the selection of ribozyme catalysts of a kind that might have facilitated polynucleotide replication. This leads to a brief outline of the Molecular Biologists' Dream, an optimistic scenario for the origin of the RNA World. In the second part of the review we point out the many unresolved problems presented by the Molecular Biologists' Dream. This in turn leads to a discussion of genetic systems simpler than RNA that might have "invented" RNA. Finally, we review studies of prebiotic membrane formation.
14787080
Self-splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening sequence of tetrahymena
In the macronuclear rRNA genes of Tetrahymena thermophila, a 413 bp intervening sequence (IVS) interrupts the 26S rRNA-coding region. A restriction fragment of the rDNA containing the IVS and portions of the adjacent rRNA sequences (exons) was inserted downstream from the lac UV5 promoter in a recombinant plasmid. Transcription of this template by purified Escherichia coli RNA polymerase in vitro produced a shortened version of the pre-rRNA, which was then deproteinized. When incubated with monovalent and divalent cations and a guanosine factor, this RNA underwent splicing. The reactions that were characterized included the precise excision of the IVS, attachment of guanosine to the 5' end of the IVS, covalent cyclization of the IVS and ligation of the exons. We conclude that splicing activity is intrinsic to the structure of the RNA, and that enzymes, small nuclear RNAs and folding of the pre-rRNA into an RNP are unnecessary for these reactions. We propose that the IVS portion of the RNA has several enzyme-like properties that enable it to break and reform phosphodiester bonds. The finding of autocatalytic rearrangements of RNA molecules has implications for the mechanism and the evolution of other reactions that involve RNA.
39111511
The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme
The RNA moieties of ribonuclease P purified from both E. coli (M1 RNA) and B. subtilis (P-RNA) can cleave tRNA precursor molecules in buffers containing either 60 mM Mg2+ or 10 mM Mg2+ plus 1 mM spermidine. The RNA acts as a true catalyst under these conditions whereas the protein moieties of the enzymes alone show no catalytic activity. However, in buffers containing 5-10 mM Mg2+ (in the absence of spermidine) both kinds of subunits are required for enzymatic activity, as shown previously. In the presence of low concentrations of Mg2+, in vitro, the RNA and protein subunits from one species can complement subunits from the other species in reconstitution experiments. When the precursor to E. coli 4.5S RNA is used as a substrate, only the enzyme complexes formed with M1 RNA from E. coli and the protein moieties from either bacterial species are active.
85861357
Autocatalytic Sets: From the Origin of Life to the Economy
The origin of life is one of the most important but also one of the most difficult problems in science. Autocatalytic sets are believed to have played an important role in the origin of life. An autocatalytic set is a collection of molecules and the chemical reactions between them, such that the set as a whole forms a functionally closed and self-sustaining system. In this article, I present an overview of recent work on the theory of autocatalytic sets and on how this theory can be used to study the probability of emergence, the overall structure, and the further evolution of such systems, both in simple mathematical models and in real chemical systems. I also describe some (still speculative) ideas of how this theory can potentially be applied to living systems in general and perhaps even to social systems such as the economy.
2010028
Serial transfer can aid the evolution of autocatalytic sets
BackgroundThe concept of an autocatalytic set of molecules has been posited theoretically and demonstrated empirically with catalytic RNA molecules. For this concept to have significance in a realistic origins-of-life scenario, it will be important to demonstrate the evolvability of such sets. Here, we employ a Gillespie algorithm to improve and expand on previous simulations of an empirical system of self-assembling RNA fragments that has the ability to spontaneously form autocatalytic networks. We specifically examine the role of serial transfer as a plausible means to allow time-dependent changes in set composition, and compare the results to equilibrium, or “batch” scenarios.ResultsWe show that the simulation model produces results that are in close agreement with the original experimental observations in terms of generating varying autocatalytic (sub)sets over time. Furthermore, the model results indicate that in a “batch” scenario the equilibrium distribution is largely determined by competition for resources and stochastic fluctuations. However, with serial transfer the system is prevented from reaching such an equilibrium state, and the dynamics are mostly determined by differences in reaction rates. This is a consistent pattern that can be repeated, or made stronger or weaker by varying the reaction rates or the duration of the transfer steps. Increasing the number of molecules in the simulation actually strengthens the potential for selection.ConclusionsThese simulations provide a more realistic emulation of wet lab conditions using self-assembling catalytic RNAs that form interaction networks. In doing so, they highlight the potential evolutionary advantage to a prebiotic scenario that involves cyclic dehydration/rehydration events. We posit that such cyclicity is a plausible means to promote evolution in primordial autocatalytic sets, which could later lead to the establishment of individual-based biology.
13370498
Required Levels of Catalysis for Emergence of Autocatalytic Sets in Models of Chemical Reaction Systems
The formation of a self-sustaining autocatalytic chemical network is a necessary but not sufficient condition for the origin of life. The question of whether such a network could form “by chance” within a sufficiently complex suite of molecules and reactions is one that we have investigated for a simple chemical reaction model based on polymer ligation and cleavage. In this paper, we extend this work in several further directions. In particular, we investigate in more detail the levels of catalysis required for a self-sustaining autocatalytic network to form. We study the size of chemical networks within which we might expect to find such an autocatalytic subset, and we extend the theoretical and computational analyses to models in which catalysis requires template matching.
4303752
Spontaneous network formation among cooperative RNA replicators
The origins of life on Earth required the establishment of self-replicating chemical systems capable of maintaining and evolving biological information. In an RNA world, single self-replicating RNAs would have faced the extreme challenge of possessing a mutation rate low enough both to sustain their own information and to compete successfully against molecular parasites with limited evolvability. Thus theoretical analyses suggest that networks of interacting molecules were more likely to develop and sustain life-like behaviour. Here we show that mixtures of RNA fragments that self-assemble into self-replicating ribozymes spontaneously form cooperative catalytic cycles and networks. We find that a specific three-membered network has highly cooperative growth dynamics. When such cooperative networks are competed directly against selfish autocatalytic cycles, the former grow faster, indicating an intrinsic ability of RNA populations to evolve greater complexity through cooperation. We can observe the evolvability of networks through in vitro selection. Our experiments highlight the advantages of cooperative behaviour even at the molecular stages of nascent life.
125147580
Autocatalytic sets and chemical organizations: modeling self-sustaining reaction networks at the origin of life
Two related but somewhat different approaches have been proposed to formalize the notion of a self-sustaining chemical reaction network. One is the notion of collectively autocatalytic sets, formalized as RAF theory, and the other is chemical organization theory. Both formalisms have been argued to be relevant to the origin of life. RAF sets and chemical organizations are defined differently, but previously some relationships between the two have been shown. Here, we refine and explore these connections in more detail. In particular, we show that so-called closed RAFs are chemical organizations, but that the converse is not necessarily true. We then introduce and apply a procedure to show how chemical organizations can be used to find all closed RAFs within any chemical reaction system. We end with a discussion of why and how closed RAFs could be important in the context of the origin and early evolution of life.
30659399
Dynamics of prebiotic RNA reproduction illuminated by chemical game theory
Significance The origins of life required a means for information-containing molecules to compete with one another for survival and reproduction. Using an analysis based on game theory, we can predict the situations in which cooperation, selfishness, or a mixture of the two is beneficial to the future evolutionary success of RNAs. Many origins-of-life scenarios depict a situation in which there are common and potentially scarce resources needed by molecules that compete for survival and reproduction. The dynamics of RNA assembly in a complex mixture of sequences is a frequency-dependent process and mimics such scenarios. By synthesizing Azoarcus ribozyme genotypes that differ in their single-nucleotide interactions with other genotypes, we can create molecules that interact among each other to reproduce. Pairwise interplays between RNAs involve both cooperation and selfishness, quantifiable in a 2 × 2 payoff matrix. We show that a simple model of differential equations based on chemical kinetics accurately predicts the outcomes of these molecular competitions using simple rate inputs into these matrices. In some cases, we find that mixtures of different RNAs reproduce much better than each RNA type alone, reflecting a molecular form of reciprocal cooperation. We also demonstrate that three RNA genotypes can stably coexist in a rock–paper–scissors analog. Our experiments suggest a new type of evolutionary game dynamics, called prelife game dynamics or chemical game dynamics. These operate without template-directed replication, illustrating how small networks of RNAs could have developed and evolved in an RNA world.
8313458
Prebiotic RNA Network Formation: A Taxonomy of Molecular Cooperation
Cooperation is essential for evolution of biological complexity. Recent work has shown game theoretic arguments, commonly used to model biological cooperation, can also illuminate the dynamics of chemical systems. Here we investigate the types of cooperation possible in a real RNA system based on the Azoarcus ribozyme, by constructing a taxonomy of possible cooperative groups. We construct a computational model of this system to investigate the features of the real system promoting cooperation. We find triplet interactions among genotypes are intrinsically biased towards cooperation due to the particular distribution of catalytic rate constants measured empirically in the real system. For other distributions cooperation is less favored. We discuss implications for understanding cooperation as a driver of complexification in the origin of life.
205973501
Prebiotic network evolution: six key parameters.
The origins of life likely required the cooperation among a set of molecular species interacting in a network. If so, then the earliest modes of evolutionary change would have been governed by the manners and mechanisms by which networks change their compositions over time. For molecular events, especially those in a pre-biological setting, these mechanisms have rarely been considered. We are only recently learning to apply the results of mathematical analyses of network dynamics to prebiotic events. Here, we attempt to forge connections between such analyses and the current state of knowledge in prebiotic chemistry. Of the many possible influences that could direct primordial network, six parameters emerge as the most influential when one considers the molecular characteristics of the best candidates for the emergence of biological information: polypeptides, RNA-like polymers, and lipids. These parameters are viable cores, connectivity kinetics, information control, scalability, resource availability, and compartmentalization. These parameters, both individually and jointly, guide the aggregate evolution of collectively autocatalytic sets. We are now in a position to translate these conclusions into a laboratory setting and test empirically the dynamics of prebiotic network evolution.
197141317
The Origin of Cellular Life
This essay presents a scenario of the origin of life that is based on analysis of biological architecture and mechanical design at the microstructural level. My thesis is that the same architectural and energetic constraints that shape cells today also guided the evolution of the first cells and that the molecular scaffolds that support solid-phase biochemistry in modern cells represent living microfossils of past life forms. This concept emerged from the discovery that cells mechanically stabilize themselves using tensegrity architecture and that these same building rules guide hierarchical self-assembly at all size scales (Sci. Amer 278:48-57;1998). When combined with other fundamental design principles (e.g., energy minimization, topological constraints, structural hierarchies, autocatalytic sets, solid-state biochemistry), tensegrity provides a physical basis to explain how atomic and molecular elements progressively self-assembled to create hierarchical structures with increasingly complex functions, including living cells that can self-reproduce.
55091807
Emergence and complex systems: The contribution of dynamic graph theory
Emergence and complex systems have been the topic of many papers and are still disputed concepts in many fields. This lack of consensus hinders the use of these concepts in practice, particularly in modelling. All definitions of emergence imply the existence of a hierarchical system: a system that can be observed, 15 measured and analysed at both macroscopic and microscopic levels. We argue that such systems are well described by mathematical graphs and, using graph theory, we propose an ontology (i.e. a set of consistent, formal concept definitions) of dynamic hierarchical systems capable of displaying emergence. Using graph theory enables formal definitions of system macro-state, micro-state and dynamic structural changes. From these definitions, we identify four major families of emergence that match existing definitions from the 20 literature. All but one depend on the relation between the observer and the system, and remind us that a major feature of most supposedly complex systems is our inability to describe them in full. The fourth definition is related to causality, in particular, to the ability of the system itself to create sources of change, independent from other external or internal sources. Feedback loops play a key role in this process. We propose that their presence is a necessary condition for a hierarchical system to be qualified as complex.
3279786
Glassy nature of hierarchical organizations
The question of why and how animal and human groups form temporarily stable hierarchical organizations has long been a great challenge from the point of quantitative interpretations. The prevailing observation/consensus is that a hierarchical social or technological structure is optimal considering a variety of aspects. Here we introduce a simple quantitative interpretation of this situation using a statistical mechanics-type approach. We look for the optimum of the efficiency function $${E}_{eff}=1/N{\sum }_{ij}{J}_{ij}{a}_{i}{a}_{j}$$Eeff=1/N∑ijJijaiaj with Jij denoting the nature of the interaction between the units i and j and ai standing for the ability of member i to contribute to the efficiency of the system. Notably, this expression for Eeff has a similar structure to that of the energy as defined for spin-glasses. Unconventionally, we assume that Jij-s can have the values 0 (no interaction), +1 and −1; furthermore, a direction is associated with each edge. The essential and novel feature of our approach is that instead of optimizing the state of the nodes of a pre-defined network, we search for extrema for given ai-s in the complex efficiency landscape by finding locally optimal network topologies for a given number of edges of the subgraphs considered.
10950909
Small-world topology of functional connectivity in randomly connected dynamical systems
Characterization of real-world complex systems increasingly involves the study of their topological structure using graph theory. Among global network properties, small-world property, consisting in existence of relatively short paths together with high clustering of the network, is one of the most discussed and studied. When dealing with coupled dynamical systems, links among units of the system are commonly quantified by a measure of pairwise statistical dependence of observed time series (functional connectivity). We argue that the functional connectivity approach leads to upwardly biased estimates of small-world characteristics (with respect to commonly used random graph models) due to partial transitivity of the accepted functional connectivity measures such as the correlation coefficient. In particular, this may lead to observation of small-world characteristics in connectivity graphs estimated from generic randomly connected dynamical systems. The ubiquity and robustness of the phenomenon are documented by an extensive parameter study of its manifestation in a multivariate linear autoregressive process, with discussion of the potential relevance for nonlinear processes and measures.