Sunday, August 18, 2013

What Came Before 'RNA World'?

I go to bed sometimes wondering what early earth was like. I try to imagine how it's possible that life could have arisen when this planet was perhaps only 1% of its current age, barely cool enough for the oceans not to boil off.

It's generally understood that life originated around 3.8 billion years ago in tide pools, swamps, lakes, or possibly the deep ocean, while organic molecules rained down from lightning-filled skies heavy with pyroclastic gases. This is the so-called Primordial Soup Theory of Haldane and Oparin, given experimental weight by Miller and Urey. It leaves open rather a lot of important details, but clearly implies that biopoiesis arose in an aqueous phase through interaction of co-solutes.

Did life begin in, under, or near hydrothermal vents?
Some researchers believe serpentinite rock structures
associated with white chimneys could have provided
pH gradients suitable for biopoesis.
From a chemical standpoint, the characteristic defining feature of life is catalysis; in particular, the catalytic formation of catalysts that catalyze their own formation. In the standard Crick dogma of DNA -> RNA -> protein, we leave undrawn the many monomer/protein interactions that lead back to DNA. Nevertheless, it's clear that 85% to 90% of proteins and 10% to 15% of RNA molecules play mainly catalytic roles in cell chemistry.

For precisely this reason, aqueous-phase Soup Theory should probably be reconsidered. Any chemist will tell you that surface catalysis and phase boundary catalysis are orders of magnitude more effective than pure liquid-phase catalysis. This is why catalytic converters on cars are not giant bongs with fluid in them but instead contain a ceramic honeycomb core overlaid with a solid-phase platinum-palladium washcoat. It is also why the largest industrial catalytic operations (including fluid catalytic cracking of petroleum oil, which is fluid only in terms of the flow of ingredients; the catalyst itself is a solid powder) employ surface catalysis. Indeed, catalysts are often used in powdered, sintered, or coated-bead form specifically to maximize surface area. In living cells, enzymes are only partially solvated (interior portions are typically hygrophobic), and most enzymes can in fact be imagined as solid fixtures onto which reactants are adsorbed. (Surely no one thinks of ribosomes as being "in solution" in the way that, say, a sodium ion is in solution.) Surface catalysis characterizes living systems as well as industrial processes.

We also know that crowding effects are important in controlling enzyme shape and activity, and in the absence of crowding, some enzymes tend to partially unfold. Indeed, it seems likely molecular confinement has (to some extent) driven the evolution of protein primary and tertiary structure. Some would argue that biological macromolecules resembling those of today could not reasonably have arisen in a confine-free aqueous phase and that (therefore) the proto-biotic "soup" envisioned by Oparinn and Haldane is unlikely to have produced cellular life. Some say it's much more likely that biopoiesis began in an environment of solvated clay particles, serpentine rock near hydrothermal vents, or (perhaps) a feldspar lattice of some kind. A colloid (such as clay) offers many advantages. For a clay to be a clay, particles must be no larger, on average, than 2 microns. This is a perfect substrate size for growth of loosely bound biological macromolecules. Such particles offer a huge amount of surface area per unit volume, much more than could be realized through, say, the attachment of catalytic foci to sheets of silica-laden rock.

Such is the state of our ignorance on biopoiesis that there's still no clear agreement on whether proteins appeared first, or nucleic acids (or perhaps biologically active lipids). The jury is still out. The so-called RNA World theory has gained a tremendous following in the last 30 years, based in part on work by Cech and Altman showing that RNA is capable of catalyzing protein formation by itself.  But a fundamental unanswered problem in RNA World theory is how pyrimidines, purines, or other monomers managed to link up with sugars and then form the first RNA molecules in the absence of a suitable catalyst. (RNA can catalyze the formation of RNA, but how did the first RNA-like oligomer arise, without a catalyst?) Pyrimidines and purines are not known to spontaneously bind to ribose, much less form phosphorylated nucleotides, on their own. By contrast, amino acids can easily condense to form dipeptides, and dipeptides can catlyze the formation of other peptides. (For example, the dipeptide histidyl-histidine has been shown to catalyze the formation of polyglycine in wet-dry cycled clay.) Thus, it's at least plausible that proteins came first.

Ironically, abiotic formation of purines and pyrimidines is not, in itself, an insurmountable problem, provided we accept that hydrogen cyanide and formaldehyde were present in the primordial "soup." (Both HCN and formaldehyde have been produced with good yields in spark-discharge experiments involving diatomic nitrogen, CO2, water, and hydrogen. Even in the absence of molecular hydrogen, the yield of HCN and H2CO can approach 2%.) HCN undergoes a base-catalyzed tetramerization reaction to produce diaminomaleonitrile (DAMN), which, with the aid of u.v. light, can go on to yield a variety of purines. Acid hydrolysis of the HCN oligomers thus produced can lead (somewhat circuitously) to pyrimidines.

Abiotic formation of sugars is also possible if formaldehyde is present. Condensation of formaldehyde in the presence of calcium carbonate or alumina yields glycoaldehyde, which can begin a cascade of aldol condensations and enolizations that produce a formidable array of trioses, tetroses, pentoses, and higher sugars via Butlerow chemistry (also called the formose reaction).

The greatest problem with RNA World theory thus isn't the ab initio creation of bases or sugars, but rather their attachment to one another. In current biologic systems, pyrimidines are attached to sugars by displacement of pyrophosphate at the sugar's C1 position (something that has not succeeded in the lab under prebiotic conditions). In living systems, purine nucleosides are created by piecing together the purine base on a preexisting ribose-5-phosphate. It's hard to see how that could occur abiotically.

It's worth noting, too, that while spontaneous creation of sugars and bases can occur through condensations and other reactions, the result would not simply be just the riboses and purines and pyrimidines seen today; rather, there would arise a zoo of different products, including all the stereoisomers of such products. (There are, among the pentoses alone, twelve different possible stereoisomers.) Somehow, early systems would have to have converged on just the sugars, just the bases, and just the isomers of them needed to promulgate living systems.

Not that an abundance of isomers is a bad thing. Maybe pre-cellular "miasmal" life actually comprised a remarkable zoo of thousands (or hundreds of thousands) of potential biomolecular precursors, of which only the most catalytogenic survived. If muds and clays offered the particle substrates on which these molecules were formed, one can imagine that sticky molecules (those with the power to adhere tenciously to clay particles, sealing them off from other, competing molecules) would have eventually won control over the means of catalysis. This would have meant micron-sized clay particles covered over with what would today be called nonsense proteins: ad-hoc polypeptides made of whatever amino acids (and other reactive species) might most easily polymerize.

What might these nonsense proteins have been capable of? In a Shakespeare-monkey typing pool world, any kind of protein is possible, subject only to steric hindrance, crowding effects, and the laws of chemistry. It seems likely that a one-micron clay particle coated with Shakespeare-monkey proteins would expose, if only by accident, hundreds of thousands of active sites of various kinds, creating catalytic opportunities of exactly the sort needed to take chemical evolution to the next stage.

Some enterprising 21st-century Urey or Miller needs to affix tens or hundreds of thousands of nonsense proteins to hundreds of thousands (or better, millions) of clay particles, soak it all in monomers of various kinds (amino acids, sugars, bases, lipids), and see what comes out. Experiments need to be done with activated colloids of various kinds, using temperature cycling as an energy source, using (and not using) oxidizing and reducing agents, with and without wet/dry cycling, with and without freezing and thawing, electrical energy, etc. We need to focus our efforts on what came before RNA World, what life was like before there were templates, before there was a genetic code, before Crick dogma. What were proteins like before the invention of the start codon or the stop codon? (Was protein size determined by Brownian dynamics? Reactant exhaustion? Molecular crowding? Intervention by chaperones or proteases?) What kinds of "protein worlds" might have existed under acidic conditions? Basic conditions? High redox-potential conditions? High or low temperature conditions? Phosphate-rich (or -poor) conditions? Repeat all of the above with and without u.v. light. With and without pyroclastic gases. With and without lightning. With and without cosmic rays. With and without adenylated coenzymes.

Experiments are waiting to be done—by the thousands—in vitro, in silico, in lutum.