The RNA World and its Role in the Emergence of Replicators

Transcription

1 The RNA World and its Role in the Emergence of Replicators The theory of evolution by natural selection has explained much in the field of biology: the diversification of organisms into species, how living things have come to look and function as though they have been designed for a purpose and the fact that all life on Earth arose from a common ancestor. Evolution results from the differential survival of replicators [1] ; an entity replicates itself imperfectly, resulting in variation in the progeny and the progeny best suited to the environment survive and replicate themselves. But how did these replicators first come about? Such a question precedes the influence of evolution and delves into the very origin of life. Consider the modern-day replicator, DNA, present in all cellular organisms. It is a large double-stranded molecule, twisted in the iconic double helix structure as first identified by Watson and Crick in What enables it to be a replicator that can evolve is the ability to store information in its nucleotide sequence and to synthesise new strands by Watson-Crick base pairing. DNA is remarkably accurate in its replication; studies in Escherichia coli have suggested a base substitution error rate in the range of 10-7 to 10-8 [2]. Clearly, DNA is a complex and stable molecule and it also has many proteins that must be present for it to function properly, such as DNA polymerase and DNA ligase. It is not likely that both DNA and its associated proteins could have arisen spontaneously from abiotic molecules and they must therefore be a product of evolution, pointing towards the idea of replicators that preceded DNA. Otherwise, the issue falls into a chicken-and-egg paradox: DNA codes for proteins (indirectly via RNA), yet proteins are required for the functioning and replication of DNA. How can this apparent cyclical dilemma be resolved? The prevailing hypothesis presented as a solution to this problem is that of the RNA world. The RNA world hypothesis states that there existed on the early Earth selfreplicating RNA molecules in place of the complex interaction between DNA, RNA and proteins. RNA, similarly to DNA, is a nucleic acid capable of storing genetic information but it is also able to catalyse reactions, like an enzymatic protein does. These catalytic RNA molecules are known as ribozymes and their discovery bolstered confidence in the hypothesis as it showed that RNA could take on both the roles of DNA and proteins. This means that RNA can hypothetically catalyse its own synthesis, leading to a self-replicating system. Although such a system with RNA as the replicator has yet to be seen in nature, there is some evidence to be found for this hypothesis when looking at the molecular biology of the modern cell. If the RNA world preceded the DNA/RNA/protein interaction, the transition may not have been complete and one may expect to find molecular fossils from the RNA world in the modern cell. And indeed, such molecular fossils exist. For example, the structure of the ribosome is often called the smoking gun of the RNA world. Ribosomes are made mostly from RNA, with proteins found only on the outside of the central RNA core; no amino acid chain comes within 18 Å of the active site for peptide bond formation [3]. When also considering the process of translation as a whole, which involves mrna, trna and rrna, it seems as though protein synthesis may have been possible without any protein at all. As Francis Crick put it, it is tempting to wonder if the primitive ribosome could have been made entirely of RNA [4]. Such evidence makes the RNA world seem plausible and although the origin of

2 protein synthesis and DNA is unknown, it can be reasonably regarded as arising from some form of selection acting on self-replicating RNA molecules. However, the question remains on how the RNA itself could have come about. Although it is simpler than DNA, RNA is still a rather complex molecule with four possible nitrogenous bases attached to a ribose-phosphate backbone. Could RNA have spontaneously formed in primordial prebiotic conditions? The answer is not clear, especially since we know very little about the actual conditions on the early Earth and how plausible certain prebiotic materials and reactions may be. The first attempt at understanding what kind of molecules may have formed in primordial times was the classic Miller-Urey experiment, published in It involved subjecting a mixture of water vapour, methane, ammonia and hydrogen (what was thought at the time to mimic Earth s primitive atmosphere) to electric discharges. What resulted was the formation of numerous amino acids, such as glycine, α-alanine and β-alanine [5]. This result was greatly influential in the support of the primordial soup theory, which proposed that simple organic molecules formed by the exposure of Earth s chemically reducing atmosphere to various energy sources, such as lightning and UV radiation, accumulated in a soup and eventually transformed into more complex polymers associated with life. However, it is now known that the experiment s assumption of a very reducing atmosphere was incorrect, so the process demonstrated is unlikely to be representative of the emergence of complex organic molecules [6]. There have been more recent attempts at finding plausible pathways by which complex molecules like RNA could have arisen. Initially, the problem of the prebiotic synthesis of ribonucleotides was approached under the assumption that the three molecular components (ribose, phosphate and the nitrogenous bases) combined together in order to assemble the ribonucleotide, having formed separately. This assumption led to a number of difficulties, especially the synthesis of nucleosides from ribose and the nucleoside bases. Orgel referred to this step as the weakest link in the chain of prebiotic reactions leading to oligonucleotides [6] and scepticism grew about the possibility of RNA arising spontaneously. Alternative replicators that could have preceded RNA were put forward, such as PNA, TNA and GNA, which are all analogues of RNA. For example, PNA is an uncharged analogue in which the backbone is held together with amide bonds and research has shown that information can be transferred from PNA to RNA in template-directed reactions [7]. This suggests that a transition from a PNA world to an RNA world would have been possible but it is unclear without further work how much easier it would have been for these pre- RNA-world replicators to come about compared to standard RNA. However, a more recent approach to ribonucleotide synthesis is the idea that the sugar and nucleobase arise from a common precursor, avoiding the use of free sugar and nucleobase molecules as intermediates [8]. Powner et al. have shown that the ribonucleotide can form from a single intermediate known as 2-aminooxazole, which forms quickly and efficiently when glycolaldehyde and cyanamide react with phosphate, which also acts as a ph buffer and catalyst [9]. The phosphate prevents the formation of many unwanted side-products but interestingly, it was also found that the majority of the by-products that are formed could be destroyed by UV radiation, while still leaving the desired ribonucleotides intact. Unfortunately, this pathway for

3 ribonucleotide synthesis yields only pyrimidine ribonucleotides, leaving the plausible formation of purine ribonucleotides unexplained. Be that as it may, the ribonucleotides must still join up in order to form RNA molecules, and there are a few problems with this step. First, nucleoside 5 - polyphosphates are high-energy phosphate esters but are not very reactive in solution. In fact, it is more probable for the hydrolysis of a polynucleotide to occur than the non-enzymatic polymerization of nucleotides, which is a very slow process [7]. However, under the influence of certain mineral catalysts such as montmorillonite, it has been shown that oligoribonucleotides as long as 55-mers could be formed from activated monomers [10]. Nevertheless, this finding does not help to address the second problem, that there must be some form of specificity in the oligomerisation to ensure that other interfering substances do not disrupt the formation of a pure oligoribonucleotide; there is no known mechanism or mineral catalyst by which useful monomers for oligoribonucleotide formation would be preferentially selected to oligomerise over the much greater number of disruptive molecules [11]. Third, the polymerization of activated nucleotides usually results in 5,5 -pyrophosphate linkages and 2,5 - phosphodiester linkages. This means that the probability that an oligomer consisting of only 3,5 -phosphodiester linkages is very slim, unless some unknown catalyst is involved. Fourth, since the nucleoside 5 -polyphosphates react so slowly in solution, not much work has been done on their non-catalysed polymerization under laboratory conditions. Instead, only indirect models using phosphoramidates as substrates exist and there is no indication that phosphoramidates, such as phosphorimidazolides, plausibly existed as prebiotic molecules [6]. Furthermore, even if a collection of oligoribonucleotides were formed in some way or another, there are still issues with the elegant idea that RNA can catalyse its own synthesis, leading to a self-replicating system. The necessary RNA replicase is optimistically thought to have to be at least nucleotides long (the minimum required to form a triple stem-loop structure) to function with reasonable efficiency [7]. Supposing that this ribozyme is 40 nucleotides long, that makes it one of possible RNA molecules of that length. However, one RNA molecule is not enough. Under the assumption that it is unlikely for the RNA replicase to catalyse the replication of itself or for a complete complementary strand to be available to the replicase automatically, the ribozyme must have another identical molecule to act upon, in order to create a copy of itself. For two such identical RNA 40-mers to arise it would require a collection of RNA molecules numbering 10 48, which Robertson and Joyce calculated to weigh roughly g; that is more than the weight of the Earth [7]. Not only does it seem highly improbable that an efficient RNA replicase ribozyme could have arisen in this way, there is also the assumption that the ribozyme catalyses the replication of only its own copies, without affecting neighbouring polynucleotides of different sequences. This specificity is necessary to ensure that only the RNA molecules able to self-replicate, and their progeny, gain a selective advantage; otherwise, all polynucleotides would benefit from the ribozyme and continue to compete with the self-replicating RNA. There is no reason to believe that an RNA replicase ribozyme would act in this necessary selective manner.

4 In this way, although the RNA world hypothesis initially seems like a neat idea, it is flawed at almost every step. From the non-enzymatic synthesis and polymerisation of nucleotides to the formation of a ribozyme capable of replicating its own copies, the hypothesis requires optimistic outlooks and the need to overcome astronomical improbabilities. Thus, the RNA-first explanation for the origin of the RNA world has been described by Joyce and Orgel as the Molecular Biologists Dream [12]. Until more research is done and the problems in prebiotic chemistry are solved, the RNA world hypothesis can only remain a Dream. So what are the alternatives? A pre-rna-world replicator hypothesis in which simpler replicator molecules invented RNA by natural selection is a possibility but as mentioned before, the proposed molecules are not much simpler and a plausible precursor system is yet to be found. A similar hypothesis that involves genetic takeover from one system to RNA is the clay hypothesis, first proposed by Cairns- Smith in the mid-1960s. It states that clay minerals were the first systems that were able to evolve by natural selection, with information coded by the irregularities in the clay crystals, such as dislocations and ion substitutions [13]. Complex organic molecules are said to have gradually arisen on the catalytic surfaces of these clays, and having invented RNA, the informational clay minerals were displaced. However, there is no experimental support for this hypothesis; in fact, studies by Kahr et al. have shown that crystals are not faithful enough to transfer information reliably between generations [14]. The most recent alternative hypothesis is that of the peptide-rna world, which argues that the close linkage between RNA and proteins we find in the modern cell today did not emerge as a result of the evolution of a self-replicating RNA system but was fundamentally essential from the very beginning. Instead of RNA having to arise on its own from the primordial soup, the hypothesis asserts that interactions between amino acids and nucleotides led to the simultaneous development of RNA and proteins. The idea first came about when Carter and Kraut showed that antiparallel polypeptides and polynucleotides adopt complementary helical confirmations [15]. This pointed towards a primitive catalytic template mechanism by which both polymers would be able to assemble together, each acting as a polymerase for the other, although this has yet to be experimentally proved. The main body of experimental evidence for this hypothesis comes with the argument that the genetic code and protein synthesis did not arise from RNA alone but by the co-evolution of peptide catalysts and RNA. The peptide-rna world hypothesis overcomes a number of problems faced by the RNA world hypothesis in this aspect. Firstly, the lack of any phylogenetic record of an RNA system transitioning to the current triplet code does not favour the RNA world. Nor is there any evidence in current biology for RNA-catalysed amino acid activation, nucleic acid synthesis or trna acylation. By contrast, Carter et al. have found putative ancestral proteins, called urzymes, by aligning multiple three-dimensional structures of members of the two aminoacyl-trna synthetase (aars) superfamilies and identifying highly conserved peptide cores [16]. These urzymes are reported to retain around 60% of the catalytic capabilities of the modern evolved synthetases, indicating that they are likely to be indeed ancestral and also may be descendants of even more primitive peptides [17]. The scarcity of activated amino acids was a great limiting factor for the

5 development of genetically coded protein synthesis, and so the ancestral peptides of these urzymes may have been involved in the evolution of translation itself. Other problems of the RNA world hypothesis addressed by the peptide-rna world include the improbability of sequence fidelity in replication due to the difficulties involved in the emergence of an effective ribozyme replicase. In the peptide-rna world, that is not an issue because stereochemical properties of the peptide-rna double helix would ensure that the correct sequences form by complementarity. Furthermore, this also solves the concern over how only 3,5 -phosphodiester linkages might form between nucleotides; only polynucleotides linked in this way would be able to form a structurally complementary helix. Despite these promising ideas, the hypothesis simply does not have enough experimental evidence to justify the Carter & Kraut model of peptide-rna reciprocal autocatalysis. In conclusion, as a solution to the question of how the first replicators came about, the RNA world hypothesis is elegantly simple in that a single molecule deals with the roles of genetic storage and catalysis but there are very many instances where optimistic assumptions must be made and improbable events must be overcome. It is true that the alternative hypotheses are not much better; there are difficulties with the formation of pre-rna replicators, clay replicators are too clumsy to encode great complexity and the peptide-rna world still requires many aspects to be backed by experimental evidence. Such obscurity is to be expected when considering the very origin of evolvable life but considering the many integral roles RNA plays in the modern cell, it seems likely that RNA had some part to play as the first good replicator. Whether peptides were involved or not from the very beginning is an issue that can only be addressed once more research has been done on the peptide-rna world.

6 References: 1. Dawkins, R. (1982). Replicators and vehicles. Current problems in sociobiology, 45, Schaaper, R. M. (1993). Base selection, proofreading, and mismatch repair during DNA replication in Escherichia coli. Journal of Biological Chemistry, 268(32), Nissen, P., Hansen, J., Ban, N., Moore, P. B., & Steitz, T. A. (2000). The structural basis of ribosome activity in peptide bond synthesis. Science, 289(5481), Crick, F. H. (1968). The origin of the genetic code. Journal of molecular biology, 38(3), Miller, S. L. (1953). A production of amino acids under possible primitive earth conditions. Science, 117(3046), Leslie E, O. (2004). Prebiotic chemistry and the origin of the RNA world. Critical reviews in biochemistry and molecular biology, 39(2), Robertson, M. P., & Joyce, G. F. (2012). The origins of the RNA world. Cold Spring Harbor perspectives in biology, 4(5), a Szostak, J. W. (2009). Origins of life: Systems chemistry on early Earth. Nature, 459(7244), Powner, M. W., Gerland, B., & Sutherland, J. D. (2009). Synthesis of activated pyrimidine ribonucleotides in prebiotically plausible conditions. Nature, 459(7244), Ferris, J. P., Hill, A. R., Liu, R., & Orgel, L. E. (1996). Synthesis of long prebiotic oligomers on mineral surfaces. Nature, 381(6577), Shapiro, R. (2000). A replicator was not involved in the origin of life. IUBMB life, 49(3), Joyce, G. F., & Orgel, L. E. (1999). 2 Prospects for Understanding the Origin of the RNA World. Cold Spring Harbor Monograph Archive, 37, Cairns-Smith, A. G., & Hartman, H. (Eds.). (1986). Clay minerals and the origin of life. CUP Archive. 14. Bullard, T., Freudenthal, J., Avagyan, S., & Kahr, B. (2007). Test of Cairns- Smith s crystals-as-genes hypothesis. Faraday discussions, 136, Carter, C. W., & Kraut, J. (1974). A proposed model for interaction of polypeptides with RNA. Proceedings of the National Academy of Sciences, 71(2), Li, L., Francklyn, C., & Carter, C. W. (2013). Aminoacylating urzymes challenge the RNA world hypothesis. Journal of Biological Chemistry, 288(37), Carter, C. W. (2015). What RNA world? why a peptide/rna partnership merits renewed experimental attention. Life, 5(1),