The emergence of primitive cells

Life on the Earth most likely arose from vast numbers of natural experiments in which various combinations of organic molecules were mixed and recombined to form complex interacting systems, then exposed to sources of energy such as light, heat, and oxidation-reduction potentials presented by donors and acceptors of electrons. This mixing and recombination probably did not occur in free solution, but rather in fluctuating environments at aqueous-mineral interfaces exposed to the atmosphere under conditions that would tend to concentrate the organic material so that reactions could occur. Through this process, incremental chemical evolution took place over a period of several hundred million years after the Earth had cooled sufficiently for water vapour to condense into oceans. At some point, membrane-bounded systems of molecules appeared that could grow and reproduce by using energy and nutrients from the environment. An observer seeing this endproduct would conclude that such systems were alive, but would be unable to pinpoint the exact time when the complex structures took on the property of life.

Here we will assume that the structures described above would be recognizable cells, and that cellular compartments were required for life to begin. This assumption differs from the conjectures of Kauffman (1993) and Wächtershäuser (1988) that life began as a series of reactions resembling metabolism. In their view, autocatalytic pathways were first established, perhaps on mineral surfaces. Over time the systems became increasingly complex to the point that self-reproducing polymers were synthesized, with cellular compartments appearing at a later stage. There is as yet little experimental evidence that allows a choice between the two perspectives of 'compartments first' or 'metabolism first'. However, because membrane structures readily self-assemble from amphiphilic compounds known to be present in mixtures of organic compounds available on the prebiotic Earth, it is highly likely that membranous compartments were among the first biologically relevant structures to appear prior to the origin of life.

Evidence from phylogenetic analysis suggests that microorganisms resembling today's bacteria were the first form of cellular life. Traces of their existence can be found in the fossil record in Australian rocks at least 3.5 billion years old. Over the intervening years between life's beginnings and now, evolution has produced bacteria which are more advanced than the first cellular life. The machinery of life has become so advanced that when researchers began subtracting genes in one of the simplest known bacterial species, they reached a limit of approximately 265-350 genes that appears to be the absolute requirement for contemporary bacterial cells (Hutchinson et al., 1999). Yet life did not spring into existence with a full complement of 300+ genes, ribosomes, membrane transport systems, metabolism, and the DNA ^ RNA ^ protein information transfer that dominates all life today. There must have been something simpler, a kind of scaffold life that was left behind in the evolutionary rubble.

Can we reproduce that scaffold? One possible approach was suggested by the RNA World concept that arose from the discovery of ribozymes, which are RNA structures having enzyme-like catalytic activities. The idea was greatly strengthened when it was discovered that the catalytic core of ribosomes is not composed of protein at the active site, but instead is composed of RNA machinery. This remarkable finding offers convincing evidence that RNA likely came first, and then was overlaid by more complex and efficient protein machinery (Hoang et al., 2004).

Another approach to discovering a scaffold is to incorporate one or a few genes into microscopic artificial vesicles to produce molecular systems that display certain features associated with life. The properties of such systems may then provide clues to the process by which life began in a natural setting of the early Earth. What would such a system do? We can answer this question by listing the steps that would be required for a microorganism to emerge as the first cellular life on the early Earth:

• Boundary membranes self-assemble from amphiphilic molecules.

• Energy is captured either from light and a pigment system, or from chemical energy, or both.

• Ion concentration gradients are produced and maintained across the membrane by an energy-dependent process. The gradient is a source of energy to drive metabolism and synthetic reactions.

• The energy is coupled to the synthesis of activated monomers, which in turn are used to make polymers.

• Macromolecules are encapsulated, yet smaller nutrient molecules can cross the membrane barrier.

• The macromolecules grow by polymerizing the nutrient molecules, using the energy of metabolism.

• Macromolecular catalysts speed the metabolic reactions and growth processes.

• The macromolecular catalysts themselves are reproduced during growth.

• Information is contained in the sequence of monomers in one set of polymers, and this set is duplicated during growth. The information is used to direct the growth of catalytic polymers.

• The membrane-bounded system of macromolecules can divide into smaller structures.

• Genetic information is passed between generations by duplicating the sequences and sharing them between daughter cells.

• Occasional errors (mutations) are made during replication or transmission of information so that a population of primitive living organisms can evolve through selection.

Looking down this list, one is struck by the complexity of even the simplest form of life. This is why it has been so difficult to 'define' life in the usual sense of a definition, i.e., boiled down to a few sentences in a dictionary. Life is a complex system that cannot be captured in a few sentences, so perhaps a list of its observed properties is the best we can ever hope to do. Given the list, one is also struck by the fact that all but one of the functions - self-reproducing polymers - have now been reconstituted individually in the laboratory. For instance, it was shown 40 years ago that lipid vesicles self-assemble into bilayer membranes that maintain ion gradients (Bangham et al., 1965). If bacteriorhodopsin is in the bilayer, light energy can be captured in the form of a proton gradient (Oesterhelt and Stoeckenius, 1973). If an ATP synthase is in the membrane, the photosynthetic system can make ATP by coupling the proton gradient to form a pyrophosphate bond between ADP and phosphate (Racker and Stoeckenius, 1974). Macromolecules such as proteins and nucleic acids can be easily encapsulated and can function in the vesicles as catalysts (Chakrabarti and Deamer, 1994) and as membrane transport agents. The system can grow by addition of lipids, and can even be made to divide by imposing shear forces, after which the vesicles grow again (Hanczyc et al., 2003). Macromolecules like RNA can grow by polymerization in the vesicle, driven by catalytic proteins (Monnard and Deamer, 2006). And finally, samples of cytoplasm from a living cell like E. coli are easily captured, including ribosomes. A micrograph of such vesicles is shown in Figure 5.2.

The ability to capture structures as large as ribosomes has led to attempts to demonstrate translation in closed vesicles. This was first achieved by Yu et al. (2001) and later by Nomura et al. (2003), who captured samples of bacterial cytoplasm from E. coli in large liposomes. The samples included ribosomes, tRNAs and the hundred or so other components required for protein synthesis. The mRNA containing the gene for green fluorescent protein (GFP) was also included in the mix, which permitted facile detection of protein synthesis. The encapsulated translation

Fig. 5.2. Ribosomes from E. coli encapsulated in phospholipid vesicles. The vesicles were reconstituted from a detergent-lipid solution in the presence of cytoplasmic extracts from the bacteria. Several ribosomes were present in each vesicle. Micrograph courtesy of Z. Martinez.

systems worked, but only a few molecules of GFP were synthesized in each vesicle, because the only amino acids available were those captured within the vesicle. This limitation was resolved by Noireaux and Libchaber (2004), who included not only the mRNA for GFP in the mix, but also a second mRNA coding for the pore-forming protein alpha hemolysin. The hemolysin produced a channel in the lipid bilayer that allowed externally added 'nutrients' in the form of amino acids and ATP to cross the membrane barrier and supply the translation process with energy and monomers (Figure 5.3). The system worked well, and GFP synthesis continued for as long as four days.

These advances permit us to consider whether it might be possible to fabricate a kind of artificial life which is reconstituted from a complete system of components isolated from microorganisms. The system would by definition overcome the one limitation described above, the lack of a self-reproducing set of polymers, because everything in the system would grow and reproduce, including the catalytic macromolecules themselves and the lipid components of the boundary membrane. However, such a system requires that the genes for the translation system (ribosomes, tRNA, and so on) for DNA replication and transcription, and for lipid synthesis must all be present in a strand of synthetic DNA. When one adds up the number of essential genes, the total is over a hundred. This might at first seem like a daunting task, but in fact it is within the realm of possibility, in that the complete set of genes required to synthesize the polio virus has been assembled in a strand of synthetic DNA (Cello et al., 2002).

Fig. 5.3. Translation in a microenvironment. In the upper figure, amino acids and ATP encapsulated in the vesicle volume are used to make small amounts of GFP and alpha hemolysin (a-HL). The membrane prevents access to external amino acids and ATP, so the system will quickly exhaust the 'nutrients' trapped in the vesicle. However, because the a-HL is a pore forming protein, it migrates to the membrane and assembles into a heptamer with a pore large enough to allow amino acids and ATP to pass into the vesicle (lower figure). The system can now synthesize significant amounts of new protein, and the vesicle begins to show fluorescence from the accumulation of GFP (Noireaux and Libchaber, 2004).

Fig. 5.3. Translation in a microenvironment. In the upper figure, amino acids and ATP encapsulated in the vesicle volume are used to make small amounts of GFP and alpha hemolysin (a-HL). The membrane prevents access to external amino acids and ATP, so the system will quickly exhaust the 'nutrients' trapped in the vesicle. However, because the a-HL is a pore forming protein, it migrates to the membrane and assembles into a heptamer with a pore large enough to allow amino acids and ATP to pass into the vesicle (lower figure). The system can now synthesize significant amounts of new protein, and the vesicle begins to show fluorescence from the accumulation of GFP (Noireaux and Libchaber, 2004).

This thought experiment clearly points out the limitations of our current understanding of the origin of cellular life. It does make clear that life could not begin as a complex molecular system of a hundred or more genes required to fabricate the simplest possible artificial cell that uses DNA, RNA, and ribosomes for self-reproduction. Instead, as noted earlier, there must have been a kind of molecular scaffold that was much simpler, yet had the capacity to evolve into the living systems we observe today. Is there any hope that we might discover such a system? One possible lead is to find a ribozyme that can grow by polymerization, in which the ribozyme copies a sequence of bases in its own structure (Johnston et al., 2001). So far, the polymerization has only copied a string of 14 nucleotides, but this is a good start. If a ribozyme system can be found that catalyses its own complete synthesis using genetic information encoded in its structure, it could rightly be claimed to have the essential properties that are lacking so far in artificial cell models: reproduction of the catalyst itself. Instead of the hundred or so genes necessary for the translation system described above, the number is reduced to just a few genes that allow the ribozyme to control its own replication, synthesize catalysts related to primitive metabolism, including synthesis of membrane components, and perhaps a pore-forming molecule that allows nutrient transport. Given such a ribozyme, it is not difficult to imagine its incorporation into a system of lipid vesicles that would have the basic properties of the living state.

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