Genesis of EdenCONTENTS
6 : RNA AND COSMOLOGY
Chemist Shows How RNA Can Be the Starting Point for Life May 14, 2009
In 1981 Francis Crick commented that 'the origin of life appears to be almost a miracle, so many are the conditions which would have to be satisfied to get it going." (Horgan 1996) Now, several findings bolster the dominant theory of genesis - that life began in an era in which RNA was both the genetic and catalytic basis - the RNA era (Gilbert 1986, Benner et.al.) in which simple replication and 'enzymatic' process based purely on RNA catalysis established evolutionary biochemistry.
The general outlines are clear. Ribose, unlike the deoxyribose in DNA, has plausible prebiotic syntheses. RNA's capacity to both form double-helices, like DNA and to also three-dimensional tertiary structures similar to proteins through base-backbone bonding to ribose fig 10(a), causes RNA to have both genetic and catalytic capacity. Simple biological RNAs have been demonstrated to have autocatalytic self-assembling capacity. The catalytic activity of polynucleotides, hinges on various forms of proton transfer fig 17(a,b,c) (Pace and Marsh 1985), in particular transesterification.
The essential core of the protein-assembling ribosome remains RNA as does the signal recognition particle which shepherds nascent proteins through the membrane. The ancient fossil nucleotide coenzymes including ATP, NAD, coenzyme-A and Vitamin B12 are all ribonucleotides. Eucaryote organisms continue to have a massive commitment to RNA processing within the nucleus, including the use of many small small nuclear ribonucleotides or snuRps involved in RNA splicing. This suggests eucaryotes have never fully transferred from an RNA-based metabolism. Reverse transcriptases also remain ubiquitous and essential for such basic functions as telomere extension, and have a common evolutionary tree, giving retrotransposons and retroviruses a potentially ancient origin in the commonality of the RNA era.
There is still debate about whether RNA was actually the primordial genetic molecule and other hybrid molecules such as peptide-nucleic acids which use peptide rather than sugar linkages also have genetic potential and plausible prebiotic status (Nelson et. al.), however it is clear RNA itself has generic status as a cosmological molecular structure on several grounds. Adenine is a principal thermodynamic product of HCN polymerization in industrial yields. All of A, G, U and C now have prebiotice status as favoured products of such reactions. Ribose is an optimal sugar conformationally in terms of permitting complementary double helix formation, and has a synthesis route from glyceraldehyde phosphate. The complementations of A-U & G-C posses a type of structural optimality among the bases. The heterocyclic polymers are restricted in their variety by the positions of N atoms required by the polymerization process. The tautomeric states of A, U, G and C indicate AU and GC may be optimal for base-pairing among close prebiotic variants.
The nucleotide unit, as exemplified in ATP consists of a direct concatenation of key products of HCN and HCHO polymerizations. Adenine and ribose are the cyclic pentamers of HCN and HCHO linked via dehydration to a dehydrating oligo-phosphate giving it the statues of a generic structure, fig 16(a) stabilized by water and Mg2+, Positive ions also play an important role in stabilizing mono- and oligo-nucleotides. It was originaly synthesized under primitive conditions by Ponnamperuma et. al. (1963). Mg2+ ions are also bound to transfer RNA and play a critical role in transesterification, balancing the negative phosphates. The fact that the polymerizing phosphodiester bond results from the removal of H2O from phosphate suggests that phosphate was the active moiety linking of the base-sugar-phosphate complex, fig 16(c) and thus drove the entire formation of nucliec acids.
RNA proved difficult for a time to induce into complementary replication in enzyme-free systems, but its relative difficulty of synthesis may be essential to its function. It is necessary that RNA be thermodynamically unstable, or life could not exist dynamically but would 'crystalize' all the way to non-genetic polymers. A variety of partial model systems of complementary replication have been realized by Orgel and his coworkers, however instabilities in polymerization have hindered experimental enzyme-free complementary polymerization of RNAs (Orgel 1992). It is clear that a regime of polynucleotide chemistry would have to have occurred stably over evolutionary time scales for an RNA-based form of life to evolve to the point where it had established translation and captured metabolic synthetic pathways.
Ferris reported (1996) that he had found a means by which the first large chains could have been forged. When his team added montmorillonite, a positively charged clay believed to be plentiful on the young Earth, to a solution of negatively charged adenine nucleotides, it spawned RNA 10-15 nucleotides long. If these chains, which cling to the surface of the clay, were then repeatedly 'fed" more nucleotides by washing them with the solution, they grew up to 55 nucleotides long. Ferris notes the clay gets RNA off the hook of having to take on the tasks of information storage and catalysis in one fell swoop. It would catalyse RNA synthesis, stocking pools with a large range of RNA strands that, as Szostak and others have shown, would evolve a catalytic capacity of their own. (Horgan 1996). Thus complementary replication can come into existence after a phase of single-stranded polymerization has given rise to a fractal RNA environment with a diverse array of oligomeric and polymeric structures, which in turn feedback autocatalytically on replication and monomer synthesis.
A central scenario out of many, including volcanic hot pools, and hydrothermal vents, is the three-phase boundary of a phosphate-rich, clay shore line under tidal or weather-related variations in a pool in which the margin is reversibly dehydrated e.g. by sun-drying. Both clays and volcanic basalts have been cited as possible mineral interfaces. Precipitated phosphate at 37o, leads to pyrophosphate formation and hence phosphate bond energy (Hermes-Lima 1990). Since the energy for nucleotide polymerization is driven by H2O removal, reversible dehydration of a medium containing phosphate, bases and sugars provides one of the most direct and simple routes to polynucleotide formation.
DIVERSE HORIZONS OF THE RNA EPOCH
A whole new field of RNA research has developed from the discovery of spontaneous splicing of RNAs in living systems by Tom Cech and the demonstrated capacity of such RNAs to function as catalysts in transesterifications and the work of Jack Szostack's teams in selective RNA catalysis (Cech 1986a). This immediately made the idea of the RNA world before proteins a natural hypothesis. This work has grown with artificial selective evolutionary studies, culminating with the development of a ribozyme which is capable of high fidelity complementary replication of short RNA oligomers of arbitrary sequence (Johnston et.al. 2001). This has become a turning point in the credibility and maturity of the RNA world as a precursor to DNA-based life which can develop as an autonomous molecular system.
The model has been extended to others for RNA-based error-correction, synthetases and the ribosome (Bass and Cech 1984, Cech 1986b, Zany and Cech 1986, Garriga et. al. 1986, Weiner and Maizels 1987). Modified ribozymes are capable of acting as polymerases which can replicate complements to subsections of themselves (Green et. al. 1990, Doudna et. al. 1991).
The discovery that RNA appears to be the agent of peptide-bond synthesis in the modern ribosome (Guthrie 1992, Pace 1992, Noller et. al. 1992) and the capacity of modified ribozymes to act as amino-acyl esterases (Picarilli et.al. 1992), the first step of ribosomal action in protein synthesis, establish RNA has the potential to act as synthetase as well as transfer, messenger and ribosomal functions. This gives RNA the capacity to act on its own to catalyse both its own replication and the ordered polymerization of proteins. Simpler model systems have also been advanced of the stereospecific capacity of D-nucleotides to act as a catalyst of L-amino acid polymerization (Lacey et. al. 1990). These results enable RNA to be the key prebiotic molecule generating ordered polynucleotide and polypeptide structures.
Szostak and Wilson (1996, Wilson and Szostak 1995) have evolved ribozymes capable of a broad class of catalytic reactions. The catalysis of previous ribozymes tended to involve only the molecules' sugar-phosphate "backbone," but these could also promote the formation of peptide bonds (which link amino acids together to form proteins) and between carbon and nitrogen. (Horgan). David Bartel a former member of Szostak's team, has evolved RNAs that are as efficient as some modern protein enzymes. The problem with most ribozymes is that they are as likely to snip an RNA molecule apart as stitch one together which makes copying a molecule fifty nucleotides long (the minimum size necessary to catalyse a chemical reaction) difficult or impossible. Bartel's new ribozymes, on the other hand, can stitch small pieces of RNA together without breaking larger molecules apart. These ribozymes use high-energy tri-phosphate bonds similar to ATP as their fuel, speeding the reaction up several million-fold. "We've got ribozymes doing the right kind of chemistry to copy long molecules" says Szostak "We haven't achieved self-replication from single nucleotides yet, but it is definitely within sight". (Cohen)
Zhang and Cech have reported a step towards the goal of linking amino-acids. They isolated RNAs that could efficiently link specific amino acids together (Zhang and Cech 1997). These pseudo-ribosomes were selected from a random pool of 1015 synthetic RNAs. They then elicited a trans-acting by coupling one of the amino acids to a short RNA with complementary sequence to the ribozyme achieving a ribozyme which whould join a ribosynthetase-amino acid to form a peptide bond with another thus relicating even more closely ribosomal function. They also found that a small region of many of the RNAs they selected was 70 per cent identical to some regions of the ribosomal RNA. "We not only copied ribosome function, we seemed to have recapitulated its evolution," says Cech. The two researchers then removed or mutated these sequences in the synthetic RNAs (Zhang and Cech 1998) any change to this region cut the activity of the RNA by a factor of between 20 and 600. This suggests this region in both the modern ribosome and the synthetic RNA may have the same role in the fusion reaction, such as holding the amino acids in the correct position and that they may have converged on the same molecular solution.
The alternative hypothesis is that life may have begun as a molecular hybrid PNA or peptide nucleic acid. PNA has a similar structure to RNA except for having a peptide backbone based on prebiotically abundant glycine and can co-instruct complementary RNA sequences and vice versa (Böhler, Nielsen and Orgel). The bases of PNA are joined together with peptide links like those in proteins which may not present the instabilities which sugars may have faced on the early earth. Matthew Levy and his colleagues (Nelson, Levy and Miller) persuaded up to 78 per cent of plausible prebiotic chemicals to transform into PNA backbone subunits amino-ethyl glycine or AEG. The acetic acid derivatives of the bases A, G U and C can likewise be generated from prebitotic reagents including NH4CN with glycine and cyanoglyceraldehyde. AEG units link up readily at 100 deg C, which may have been common temperature four billion years ago when our planet was rich in volcanic activity. PNA is clearly an alternative route to establishing the RNA era which also has a good cosmological foundation.
UNIVERSAL STABILITY STRUCTURES IN MOLECULAR BIOLOGY
The previous discussion of the RNA era can unravel a double-bind that is central to biogenesis - how did the core biochemical pathways become generated? The traditional viewpoint is that they were successively created starting from a simple chemical-feeding heterotroph, through mutational evolution, building one-by-one the protein components necessary to make a working whole. This however does not explain how integrated systems such as electron transport and the citric acid cycle could have functioned at all with only a vestigial complement of enzymes.
This suggests that many of the major features of molecular biology are generic structures which can come into existence under suitable conditions, through bifurcation, independently of the emergence of genetic RNA, and that these were subsequently captured by genetic takeover as genetic complexity permitted. Such generic structures include the polymeric structure of proteins and nucleic acids, nucleotide coenzymes, bilayer membrane structure and the topological closure of the cell, ion transport and membrane excitability, membrane-bound electron transport, glycolysis and the citric acid cycle.
Such a perspective has far-reaching consequences for molecular biology in cosmological terms, for while the details of mutational evolution will be unique to each environment, the major features underlying biology could be universal.
(a) Nucleotides and the Nucleotide Coenzymes. The nucleotide co-enzymes are widely regarded as ancient molecular fossils retained from the RNA-era. In addition to the key role of ADP and ATP as energy currency in the bio-metabolism, GTP is used in protein synthesis, and the nucleotides UDP and CDP are carriers of glucose and choline and other membrane components. Model prebiotic reactions have successfully coupled UDP and CDP to glucose and choline (Mar et. al. 1986). Both NAD, and FAD function as carriers of redox energy. Coenzyme A consists of adenosine coupled to pantothenic acid and functions as a carrier of acyl and other groups via the terminal SH bond (Reanney 1977). Vitamin B12 also illustrates how a di-nucleotide can bind a metallic porphyrin ring. Eschenmoser (1988) has also discovered a plausible prebiotic pathway generating the more complex B12 molecule which involves two nucleotides and a Co-porphyrin.Prebiotically such a molecule could have also utilized a lowered Fe2+- Fe3+ activation energy as a carrier of electrons.
(b) Translation. According to the genetic takeover hypothesis, evolution of RNA captured existing stability structures in the prebiotic medium. The most central of these is clearly the use of proteins as coded enzyme catalysts. Such a process could only have occurred in an environment in which RNAs coexisted with amino-acids and in which a very small additional genetic advantage could capitalize on simple coding of existing structures to good effect.
A variety of amino acids and oligopeptides are common products of prebiotic syntheses. The polymerization of amino acids and the development of peptide backbones with cyanide side chains from the linear HCN oligomer fig 14, provide alternative routes to oligopeptide structure. A natural propensity for -NH2 and -CO2H moieties as basic and acidic groups arises directly from the electronegativity bifurcation.
The discovery that ribosomal, synthetase, messenger and transfer functions of protein synthesis can all in principle be carried out by RNAs alone leads to a natural interpretation of the development of the genetic code from a protein-free translation system. The major partitions of the genetic code have structural features consistent with an origin in underlying chemical bifurcations. The fundamental bifurcation sequence, fig 18 is as follows:
1 Polarity bifurcation: There is a major bifurcation in polarity between amino acids with anticodons having centre bases U & A. Uracil is correspondingly more hydrophilic than adenine, as reflected in their dominant split in hydrophobicity A(3.86)>G(2.3)>C(1.5)>U(1.45) and water solubilities A=1/1086, U=1/280. This leads to the idea that the polarity bifurcation was a principal symmetry-breaking factor in the origin of the nucleic acid code (King 1982).
2 Abundance and GC: The initial base G also codes the most abundant amino acids, consistent with a GXY code starting with GAY=polar (anticodon U), GUY=non-polar (anticodon A) providing binding strength of GC and frame shift suppression (Y=pyrimidine).
3 Four-fold code: Extending to include GGY, GCY, provides a fourfold specificity for polar (Asp/Glu), non-polar (Val and larger), along with Gly, and Ala as most abundant.
4 Eight- and Twelve- fold codes: This could have then doubled to and 8-word code by including CAY, CUY, CGY, and CCY coding for non-polar and basic groups, and then a similar series based on AAY, AUY, AGY, and ACY Wong (1975) originally noted a correspondence between the first codon base and biosynthetic pathways in primitive organisms such as sulphur bacteria with Pro, Arg, Gln Leu, His derived from Glu and having first codon base C and Ser, Thr, Ile, Asn, Met, Lys being derived from Asp having first codon base A (Knight, Freeland and Landweber 1999). OH- and SH-containing amino acids also form a single additional block (UA)(GC)Y, suggesting a third bifurcation for H-bonding, with UAY reading stop. Notably these is significan stereospecific affinity between certain amino acids such as Ile and Arg and their codons (ibid).
5 Evolutionary takeover: From this point evolutionary selection begins to optimize the bifurcations caused by stereospecificity and the growth of these interactions into synthesis pathways, based on error minimization and the incorporation of the last of the amino acids. Later assignments such as Trp are consistent with evolutionary adaptions.
Freeland and Hurst (1998), have shown that strong selective
pressures must have acted on the code during its evolution. Hurst
found that single-letter changes to a codon, inserting the wrong
amino acid into a protein, tended to specify amino acids that
were very similar chemically to the correct ones, minimising the
impact on the protein. Freeland then reasoned that the code should
minimise chemical differences most between the correct and incorrect
amino acid at the third base in the codon since translation misreads
this base 10 times as often as the second. In an analysis that
gave extra mathematical weight to the vulnerable sites most likely
to be mistranslated, Freeland showed that no more than one in
a million random codes was better at reducing the impact of errors
than the natural code. The possibility of evolutionarty change
in the code is affirmed by both mitochondrial and nuclear variants
(Knight, Freeland and Landweber 1999).
Following on from this Freeland et. al. (2000) have analysed other
work showing that more optimal global solutions do exist to propose
that stereochemical and synthesis path constraints fixated the
code ealy on into one which was later evolutionarily optimized
on error minimization constraints, the modern code being optimal
under these constraining conditions. This analysis gives strong
weight to the idea that the form of the code is derived from chemical,
historical and selective factors rather than being a frozen accident
which happened to the predecessors of the last common ancestor
of living cell lines.
(c) The Membrane, Excitability and Ion Transport :
All life as we know it is dependent on maintaining a distinct
internal micro-environment as an open far-from-equilibrium thermodynamic
system (Glansdorff and Prigogine), through the topological closure
of the cell. Viruses for example all depend on cellular life.
The structure of the bilayer membrane is a direct consequence
of the polarity bifurcation. The formation of amphophilic lipid-like
molecules, joining a linear non-polar hydrocarbon section to an
ionic or H-bonding polar terminal, leaves 2 degrees of freedom
for layer formation. Backing of the non-polar moeties to one another,
fig 20(b), completes the bilayer. Cell structure can then arise
directly from budding of the bilayer, as illustrated in budding
in several types of prebiotic reaction medium. Microcellular structures
are abundant in many origin of life syntheses, fig 19. The use
of CDP associated with choline, inisotol & lipids in membrane
construction is consistent with membrane formation in the RNA
era. The structure of typical biological lipids such as phosphatidyl
choline display a modular structure similar to ATP, consisting
of fatty acid, glycerol, and substituted amine again linked by
dehydration and involving phosphate, fig 20(e).
The existence of the membrane as a non-polar structure leads to segregation into ionic and non-polar reaction phases. Ion transport is essential in maintaining the concentration gradients that distinguish the cytoplasm from the external environment and thus must develop in the earliest cellular systems (MacElroy et. al. 1989). Ion transport is a source of significant electronic effects, because the membrane under polarization is piezo-electric and is capable of excitation in the presence of suitable ions. Model systems using the simple 19 unit oligopeptide Na-ionopore alamethicin and artificial membranes display action potentials (Mueller and Rudin 1968). Similar results have been reported for microcells produced by prebiotic techniques containing light irradiated chromophores (Przybylski and Fox 1986), demonstrating that such effects are fundamental to the quantum architecture of lipid membranes (King 1990). Four groups of non-polypeptide neurotransmitters: acetyl-choline, catecholamines, serotonin and histamine are amines, the latter three being derived from amino acids tyrosine, tryptophan and histidine by decarboxylation. Two others are amino acids and thus also contain amine groups. Notably alamethicin also has glutamine amides located in the core of the pore (Fox & Richards 1982). The catecholamines are linked to indoles such as serotonin by a prebiotic pathway, fig 20(c).
(d) Electron Transport
The fact that the proton is soluble in water to form the hydrogen
ion, but the electron is not, unless attached to another group
such as a protein, causes a physical linkage to exist between
the polarity bifurcation and the charge bifurcations associated
with electron and proton transfer, fig 20(b) mediated by H transport
through quinone reduction, (c). Despite the complexity of modern
electron transport in photosynthesis and respiration, there is
considerable evidence that membrane electrochemistry could have
arisen before translation could produce coded enzymes. Firstly
there is a consistent basis for the existence of many of the components
of electron transport during the RNA era, since the nucleotide
coenzymes NAD, FAD, a nucleotide-bound Mg & Fe-porphyrin ring
similar to B12, a cysteine-bound FeS group (Hall et. al. 1974),
possibly based on glutathione (g-glutamyl-cysteinyl-glycine) and
quinones provide all the key components of electron transport
in an RNA dependent but protein-free form, fig 20(d) (King 1990).
Both porphyrins and quinones have obvious prebiotic syntheses
and the primal role of nucleotide coenzymes has already been discussed.
Secondly, membrane structure and the solubility differences between
the electron and proton guarantee a link between electron and
hydrogen ion transport fundamental to quantum symmetry-breaking.
Electron transfer does not require the complex coded active sites
required to catalyse specific molecular transformations. Model
systems using Fe-porphyrins and imidazole can couple oxidative
electron transport to phosphorylation (Brinigar et. al. 1966)
and photo activated Mg-porphyrin to phosphate link (Goncharova
and Goldfelt 1990, Lozovaya et. al. 1990).
(e) Glycolysis
forms a bridge between six and three carbon sugars, reversing
the structural pathway from H2CO, glycoaldehyde and glycderaldehyde
to cyclic sugars, fig 15(b). Glycolysis is made energetically
possible by phosphorylation, and releases high energy phosphate
capable of driving other phosphorylations (Hermes-Lima and Vieyra
1989), fig 21(a). It is notable that glycolytic di-phosphorylation
of fructose is homologous with the route for nucleotide formation
of fig 16(c). The high phosphate environment leading to RNAs would
then naturally lead to similar phosphorylation of other sugars,
and release of the high-energy phosphate bond through cleavage
of the sugar. Mineral catalysis associated with phosphate gives
the glycolytic pathway a natural basis for lysis of sugars as
a dissipative structure. Bological UDP-glucose coupling is consistent
with nucleotide-dependent glycolysis in the RNA era.
(f) The Tricarboxylic Acid Cycle :
forms a pool of multiply carboxylated molecules which carry
CO2 in various states of energy, and result in reducing energy
via nucleotide coenzymes NAD and FAD, which coupled with the use
coenzyme A provide a basis for the tricarboxylic acid cycle in
the RNA era. This could have existed as a limit cycle of di- and
tri-carboxylated molecules acting both as an acceptor of acetate
(a carbohydrate-equivalent i.e. (H2CO)2) and as an emitter of
molecular CO2 and reducing H, thus bifurcating carbohydrate level
redox potential into reduced and oxidized components.
The linkage to nucleotide coenzymes such as NAD would have served to create a bifurcation of redox potential in the molecular milieu contributing to the diversity of reacting species. The cycle may have been hypercyclic (Eigen et. al. 1981) or chaotic, consisting of a population of molecules undergoing various generic transformations with net inflow of carboxylic acids and net emission of CO2 and transfer of H, due to generic transformations as illustrated in fig 21(b). Isomerization would have been catalysed by Fe2+. Several steps may have been driven by sunlight photolysis (Waddell et. al. 1989).
The probability that the the central structures of molecular biology existed in the RNA era is consistent with their being chemical stability structures utilized by catalytic RNAs. The small genomes during the RNA era and limited catalytic capacity of RNAs by comparison with protein makes it likely that the emerging RNA-based system had to capitalize on existing chemical stability structures becase it lacked enzyme-based biosynthetic pathways. Genetic takeover also places these stability structures in a category determined by the cosmological milieu, thus giving evolutionary biology a cosmological foundation.
THE PRECOCIOUS ORIGINS OF LIFE ON EARTH
Far from being an improbable accident taking billions of years to find the right conditions, life appears to have become established on Earth as soon as the conditions permitted a liquid water ocean, suggesting either that Earth was richly bombarded with complex organic molecules which quickly found within the diversity of microclimates on Earth some which were directly conducive to the processes leading the to the genetic epoch, or that life's had already begun in the gas and dust cloud initially forming the solar system. Gustaf Arrhenius, (Mojzsis et. al.) studying tiny apatite grains in the Isua formation of Greenland, has found carbon 12 to 13 ratios consistent with the grains originating from living matter. The Isua rocks date from 3.85 billion years ago. Although indications from zircon crystals indicate a solid crust 4.2 billion years ago, no intact rocks have been discovered older than 3.96 billion years. The moon and probably the Earth likewise was heavily bombarded with meteors up to 3.8 billion years ago, suggesting that life evolved on earth as soon as environmental conditions allowed.
The origins of the first fossil life forms including the stromatolites in fig 23, likewise lie at the limits of the geological record. At 3.5 billion years old, fossilised bacteria are the earliest evidence of life on Earth, and yet these relics, with names like Chromoccoceae and Oscdlotorioceoe, are identical to the sophisticated modern cyano-bacteria that cover the globe from Antarctica to the Sahara (Let There be Life New Scientist 6 July 96).
The emergence of the eucaryotes that lead to the higher organisms is also almost as ancient as the geological record. Traces of oil extracted from Australian shale have pushed the date for the origin of complex cells back another half a billion years. Compounds in the oil suggest that eucaryotic cells, which make up all life on Earth except for bacteria, had evolved as early as 2.7 billion years ago. It is not until about 2.1 billion years ago that fossil imprints appear in the geological record that are so large that they can only be eucaryotes. A team of researchers in Australia has found steranes, molecules with 26 to 30 carbon atoms arranged in four rings, in droplets of oil extracted from rock 700 metres below the surface in the Pilbara region of north-western Australia. These are produced by the decay of cholesterol and other steroids found in the membranes of eukaryotes, but not bacteria (Brocks et. al.). Genetic analysis of the base of the tree of life indicates the oldest branches of both archaea and (eu)bacteria are thermophilic suggesting a period in hot pools or significant meteoric impact leaving only the thermophiles as survivors.
