Origin of Life - Self-assembly, Self-organization, and Emergent Phenomena

Contents

Origin of Life

concepts of self-assembly, self-organization and the phenomena of emergence (from the prebiotic soup, Figure 01). The explanations are all based on the science of physics and chemistry although ultimately life could be better described at the level of biology. Giving enough time, life is inevitable provided the condition is right. For example, the hydrogen bond so important for maintaining the structure of life will be disrupted by thermal

Figure 01 Origin of Life [view large image]

agitation at few tens of degrees above room temperature; and the structure becomes frozen if temperature is low enough for the van der Waals force to take over.

Self-assembly

Order - The self-assembled structure have a higher order (lower entropy) than the isolated components. According to the second law of thermodynamics its surrounding would become more disorder (higher entropy). Interaction - It usually involves only the weaker hydrogen bond, which makes the structure more flexible enabling the system to withstand minor perturbation (the ability of reversibility). Building blocks - They are not only atoms and molecules, but span a wide range of nano- and mesoscopic structures, with different chemical compositions, shapes and functionalities. Examples include crystals, colloids, lipid bi-layers. The folding of poly-peptide chains into proteins and nucleic acids into their functional forms are examples of self-assembled biological structures.

Figure 02 Self-assembly [view large image]

Figure 02 shows some examples of self-assembly from peptide components to nano-structures. The alphabet in there represent the individual amino acid.

Self-organization

is non-linear similar to chaos. Another major difference is the interaction. While the interaction in self-assembly is more elementary, which can be traced back to the electromagnetic force; the interaction in self-organization means the kind of action that affects many sides. The size of the resulting structure from self-assembly is mostly mesoscopic, but it can be considerably larger for self-organization. Figure 03 illustrates the continuum of open thermodynamic systems from ordered, near-to-chaotic, to far-from-equilibrium states. As energy consumption increases, systems move further from equilibrium and pass

Figure 03 Self-organization [view large image]

through a phase transition between order and chaos. Complex systems such as life exist in this phase transition. The diagram also shows the creation of life from a cell and its individual parts (the organelles).

Emergent Phenomena

Self-organization is different from emergent phenomena. While both are dynamic processes arising over time, emergence emphasises the presence of a novel coherent macro-level emergent (property, behaviour, structure, ...) as a result from the interactions between micro-level parts (rectangles in Figure 04b). Self-organisation emphasises the spontaneous increase in order (circle in Figure 04a). When both the self-organizing and

Figure 04 Three Auto-systems

emergent processes are involved in creating the system (as in Figure 04c), it is often mistaken that they are the same process. It is this kind of process that is most important for the origin of life.

	correspondence offer an example of emergent phenomena without self-organization - it is a change of perspective from individual molecules to thermodynamics without any change in the entropy of the system. The example of slime moulds life cycle is for the case of simultaneous
Figure 05 Examples of Auto-systems [view large image]	occurrence of self-organization and emergent phenomena (Figure 05).

	The self-organization and emergent phenomena occur simultaneously when the slime moulds (see more in "Slime Moulds Life Cycle") run out of food. As shown in Figure 06, The parts are the individual amoebae, the interaction is the adhesive molecules, the energy is supplied by their favorite food - the bacteria and yeast, and the emergent phenomena is the moving slug
Figure 06 Slime Mould [view large image]	and fruiting body.

In summary, the minimum requirements for emergent biologic phenomena out of self-organization include :


1. There are interactions between components parts to build the structure.
2. An open thermodynamic system with many parts to allow the infusion of energy and removal of entropy.
3. A positive feedback loop causing nonlinear dynamic behavior (for amplifying small fluctuations).

It follows that the resulting characteristics of the biological emergent phenomena are :


1. Novel properties not observed previously in those component parts - the total is more than the sum (via interaction).
2. The resulting entity is coherent and could maintain itself as a whole over some period of time (by energy infusion).
3. It can evolve to something else. This is the Darwinian selection, which determines the outcome of the evolution according to changing environment (in positive or negative feedback loop).

A 2018 Update on the Theory of "Prebiotic World"

Evolution of Ribosome : starting from recent observations to some evidences from the ancient world.

Most theories on the origin of life adopt a "bottom-up" approach from simple to complex and from unknown to known. Another method is to trace the evolutionary path from a known point at present. The path is narrowing down by observing a conversed organelle called ribosome inside all life including bacteria and human. Although difference exists on the ribosome across different species, there is a core structure inside that is present in all of them. The following is an attempt to reach the origin by such scenario from "The Modern Day Ribosome" (structure and function) to "History of Ribosome" (looking backward to LUCA).

Figure 07 RNA World [view large image]

Figure 07 portrays one of the bottom-up theories - the hypothetical "RNA World" in a evolutionary sequence from some nucleotides ~ 4.5 Gyr ago to LUCA (Last Universal Common Ancestor) ~ one billion years later.

• The Modern Day Ribosome (structure and function) - The main function of the ribosome is to make protein (folded 3D version of the peptide chain, Figure 08a) according to the genetic codes (each codon = 3 nucleotides) on the mRNA. There is an intermediary in the form of tRNA, which associates every anti-codon with an amino acid (see Figure 08). The ribosome is composed of a small (SSU) and a large (LSU) subunits each one is a mixture of rRNA and proteins (Figure 09). In the absence of mRNA, all these components have its own existence separating from each others (Figure 10). Mammalian cell contains about 100 million of the ribosomes [either free or bound to the rough endoplasmic reticulum (ER) for exporting the protein outside the cell] and about equal number of tRNA. As a very good example of "Self-Assembly" in action,

  	each one of these parts assembles into a working ribosome at the presence of mRNA. The generation of peptide chain occurs at the active site, where the rRNA in the LSU acts like an enzyme (ribozyme, aka peptidyl transferase) to add another amino acid from the tRNA at A-site to the growing peptide chain at P-site (Figure 10). [view large image, 08a].      In chemical formula :
  Figure 08 Genetic Code / Amino Acids / Peptide Chain [view large image]	(peptide chain)n + ribosome + mRNA + (tRNA + amino acid) + ATP + H2O      (peptide chain)n+1 + ribosome + mRNA + tRNA + AMP + PPi + 0.6 ev

  See Footnote on ATP

  			The aggregate also recruits proteins called elongation factors (EF) to transfer the tRNA into the ribosome at A-site and to push the mRNA to the P-site for adding another amino acid to the growing peptide chain. The action is self explanatory by Figures 10, 11; while the video in Figure 11 further articulates the
  Figure 09 Ribosome Structure [view large image]	Figure 10 Ribosome and tRNA in Action [view large image]	Figure 11 rRNA Translation [view animation]	processing sequence which moves the whole thing to an ER at the end.

  
  Polymerase is an enzyme for synthesizing long chains of nucleic acids of either DNA or RNA from genes in the DNA. In particular, the RNA polymerase I (Pol I) synthesizes the rRNA in LSU and SSU, while Pol III makes tRNA, and the very short 5S rRNA in the LSU. Pol II produces a precursor of mRNA, which (depending on species) would synthesize 50 - 80 individual proteins that provide support for ribosome assembly and translation from mRNA to peptide chain (Figure 12).
  

  		Figure 13 shows the common core of the bacteria SSU rRNA in the form of helices (single RNA strand folds back to form double helix) labeled by numerals from 7 to 10 (see Figure 13,A) with a total length of 92 nt (nucleotide). The difference to this basic form in other species occurs mostly from the extension of helix 9 (shown in different color in different species). Figure 13,B is the 3D views, 13,C the 3D superimposition, 13,D shows the region of insertion (fingerprint). Thus, the common core can be considered as the basic structure of the SSU rRNA soon after the emergence of life. There is similarly a basic core for the LSU rRNA.
  Figure 12 Ribosome Assembly [view large image]	Figure 13 Ribosome Variation	see 3-D Pol III and a video on "cryo-EM" (for making such image).



• History of Ribosome (looking backward to LUCA) - Following the investigation of rRNA structures across the tree of life, a model based on observations categorizes the "Pre-LUCA History" into 6 phases as summarized below from modern to ancient (Figure 14) :
  
  • Phase 8 (not shown in Figure 14) - This phase marks expansions in metazoan (multicelluar organism) ribosome. The additions serve as docking sites for chaperones (escort), and regulatory proteins.
  • Phase 7 (not shown in Figure 14) - The rRNA develops eukaryotic-specific expansions which are stabilized by eukaryotic-specific proteins.
  • Phase 6 - The rRNA common core is finalized, and its genetic code is optimized. The ribosomal surface is an integrated patchwork of rRNA and mature ribosomal proteins. Many expansions acquired in this phase serve as binding sites for ribosomal proteins. The interaction between the two subunits is further elaborated by bridges involving both rRNA expansions and ribosomal proteins. Other expansions form the translocation stalk from P-site to E-site. The exit tunnel for the growing peptide chain is still under development. LUCA emerges at the end of this phase.
  • Phase 5 - Ribosomal evolution from this phase forward (to 6, 7, 8) is a tight collaboration between RNA and protein. The ribosome achieves primitive decoding ability.
  • LSU - It acquires the ratcheting system, which moves the mRNA along. The binding sites for elongation factors (EF) G and Tu are established. The 5S rRNA now associates with and reinforces the central protuberance (CP, see Figure 09). The peptide chain exit tunnel is extended. The LSU joints the SSU from each side by bridges.
  • SSU - The P-site and A-site is stabilized. There is expansion for decoding, translocation, and proof reading. Intrinsic flexibility incorporated into the SSU allows the domains (distinct structures) to move relative to each other, providing a mechanical basis for translocation.
  • tRNA and mRNA - Anti-codons in tRNA form specific interactions with mRNA codons. mRNA is polymeric with a defined sequence. The genetic code begins to optimize and expand.

  Phase 4 - The ribosome is a noncoding, diffusive ribozyme. The LSU, the SSU, proto-mRNA, and tRNA can be coupled and evolves together. The subunit interface is well developed, with numerous RNA-RNA interactions. In the LSU, the tunnel is further elongated and rigidified, facilitating the production of longer condensation products. LSU - It gains mass and assumes a hemispheric shape in correlated evolution with the SSU. The exit tunnel is extended. SSU - Newly acquired rRNA creates well-defined binding pockets for tRNAs. Primitive binding sites for elongation factors (EF) G and Tu are formed by expansion. tRNA and mRNA - tRNAs are optimized to form quasi-stable triplets matched to the proto-mRNA, which remains a single-stranded oligomers. Phase 3 - Correlations in the evolution of the LSU, SSU, tRNA, and mRNA are initiated. Catalytic efficiency and product length are increased. LSU -The PTC (Peptidyl-Transferase Center) is encased, and the exit pore is extended into a short tunnel by expansion. An embryonic subunit interface is formed. SSU - The evolution is uncorrelated, but it docks into the bridge between the subunits correctly. tRNA and mRNA - The LSU, tRNAs, proto-mRNA, and the SSU form a noncovalent quaternary complex. Proto-mRNA is a random population of single-stranded oligomers. Phase 2 - The evolution and function of the LSU and SSU are uncorrelated. The LSU is a crude ribozyme, catalyzing nonspecific, non-coding condensation of amino acids. The exit tunnel (just a pore at this stage) retains and stabilizes reaction intermediates, facilitating the production of oligomers. Charged CCA tails (Figure 10) in tRNA, which is in the form of minihelices, deliver activated substrates to the PTC. SSU function may involve association with single-stranded RNA. The RNAs grow by expansions. LSU - The PTC is stabilized, exit pore formed. Expansions develop for the A-site and P-site. SSU - The initial expansions produce a quasi-stable fold. tRNA - Proto-tRNA obtains a CCA tail. It also acquires the amino acid acceptor stem and then the T-stem and T-loop to form a minihelix (a special recognition site for the ribosome to form a tRNA-ribosome complex) . Phase 1 - The LSU and SSU start to evolve uncorrelated from ancestral RNA and primordial stem-loops. These defect-laden loops are stabilized by interactions with metal cations [metal (metal + + 1 free e-)] and the folding protects them against chemical degradation.

  Figure 14 Riobsome Evolution [view large image]

  See original article : "History of the Ribosome and the Origin of Translation".

  
  

Evolution of aaRS : the "Protein/RNA World"




		The model above is essentially the theory of RNA world looking backward. It postulates the RNA to perform both the replicative and catalytic functions. While catalyst can be made from RNA folding, RNA self-replication is difficult to create in laboratory. One suggestion is by ribozyme catalyses as shown in Figure 07,
Figure 15 RNA Replication by Heating and Cooling	Figure 16 Protein/RNA World [view large image]	Another theory considers heat separation of the RNA minihelix and piecemeal assembly as shown in Figure 15.

The alternate is the "Protein/RNA World", which is devised on the following considerations :


• Failure to self-replicate RNA without enzyme in laboratory (see "Before RNA World").
• The catalytic properties of ribozymes are not sufficient to generate RNA replication as observed so far.
• Contemporary proteins are assembled by a ribozyme (~ribosome) and contemporary nucleic acids are made by a protein polymerase.
• None of the biological reactions (called metabolism in such context) would take place if the participating substances are merely mixed together. It requires catalysts (called enzyme for making complex organic molecules) to speed up the reaction (see Figure 30) and other kinds of proteins to self-assemble everything into a functional unit.
• Amino acids would have been as abundant as the nucleic acid bases in the prebiotic world (see "prebiotic chemistry"). They could as easily be combined into peptides (proteins) to serve as catalysts.
• The requisite specificity is low in the initial stages of the Protein/RNA world, but unacceptably high in the RNA world. Low specificity processes occur with greater frequency and hence are more likely to have occurred first.
• Experiment has demonstrated that the two distinct aminoacyl-tRNA synthetase (aaRS, see Figure 17) to link up the tRNA with amino acid are, very probably related to one another. They arose as products of sense/antisense translation by processing from opposite ends of a single gene. These two classes of aaRS still retain some small, highly conserved fragments of the urzymes (ancestral enzymes), which contain two domains for catalytic and anticodon-binding functions respectively.

Here's a summary of the evolution of the Protein/RNA World (forward in time) with the development of the aminoacyl-tRNA synthetase (aaRS) playing the central role as illustrated in Figure 16 (see more in "Peptide/RNA Partnership") :


1. Biology emerged from a reciprocal partnership in which small ancestral oligopeptides and oligonucleotides initially both contributed rudimentary information coding and catalytic rate accelerations.
2. The superior information-bearing qualities of RNA and the superior catalytic potential of proteins emerged from such complexes only with the gradual invention of the genetic code.
3. The two aminoacyl-tRNA synthetase (aaRS) ATP binding sites (including a 46-amino acid sequence - the 46mers, see Figure 17 for location of the site) arose as translation products from opposite strands of a single gene. This is the key intermediates on a path from simple catalytic peptide/RNA complexes to contemporary aminoacyl-tRNA synthetases. This scenario provides a path to increasing complexity that obviates the need for a single polymer such as the RNA to act both catalytically and as an informational molecule.
4. The pattern persisted until the evolution of the modern aminoacyl-tRNA synthetases (aaRS), tRNAs, and ribosomes enabled higher-specificity genetic coding. The LUCA emerged at this stage. The early steep raise of the evolutionary curve in Figure 16 indicates relatively easy steps in synthesizing macromolecules such as RNA and protiens, while the nearly flat portion of the curve later signifies the more difficult task of making them to perform useful functions such as replication of a strand of RNA. That is, synthesis is relatively easy by just adding another unit randomly selected, while replication involves one to one copying of a specific sequence.

is only implied in the formation of the proto-CCA (Figure 10). The RNA World follows the evolution of ribosome to arrive at the ancestral rRNA. While the Protein/RNA World as portrayed in Figure 16 emphasizes the role played by aaRS, which bears evidence of ancestral protein (enzyme) in the very beginning of the evolution of life. These two theories are complementary to each other. Evolution on both ribosome and aaRS should be considered in any future investigation into the "Origin of Life". For example, there would be no translation by the ribosome without the tRNA charged by the aaRS. See "Origin of Translation" for more info on aaRS in action.

Figure 17 aaRS in Action [view large image]

BTW, most cells contain 20 different aaRS enzymes, one for each amino acid. The editing function of the aaRS as indicated in Figure 17 is to ensure high fidelity of tRNA charging.

Requirements for the Origin of Life : Self-assembly, Physics and Chemistry

• Self-assembly of RNA Virus - Most of the time a virus just lies dormant as an inert crystal, lifeless as a rock and perhaps staying that way for centuries or millenniums. Yet, unlike a rock, it may wake up at any moment, when there is the warmth and moisture of some vulnerable cell that it can swiftly enter and infect. Figure 18 shows the replication process for the RNA virus.

  After entering the cell by endocytosis, the virus becomes uncoated. The DNA then replicates more of its kind and simultaneously making new coating proteins. These parts assemble to form more viruses, which exit from the host to infect more cells. One of the theories proposes that viruses originated and evolved along with the most primitive cells that first contained self-replicating abilities. Irrespective of the various hypotheses, the fact that the viral parts re-pack themselves, constitutes the strongest support for the role of self-organization and emergent phenomena in the origin of life. This case involves the self-assembly of macromolecules such as RNA, and proteins (the parts) inside the host. The self-organization also occurs within the host at its expense. Finally, a new virus (a coherent whole entity) appears with emergent property which can infect another cell. This is everyday occurrence in modern time. The origin of life would be a step further back when the viral genes and proteins are self-assembled in the primordial soup. It took about one billion years for such assembly to emerge on Earth (Figure 01).

  Figure 18 RNA Virus [view large image]

  The followings provides further details on RNA virus replication in 7 steps (Figure 18) :

  
  
  There are numerous kinds of viruses, this summary only considers the single-stranded, positive-sense (its RNA is the same as mRNA) RNA virus (such as the Coronaviridae), which replicates solely in cytoplasm of the host cell.
  
  
  1. Adsorption - Viruses can infect only certain species of hosts and only certain cells within that host. Cells that a virus may use to replicate are called permissive. It is the special kind of membrane receptor that allows certain kind of virus to enter. The permissive cell must also possess the materials required by the virus to replication.
  2. Entry - A channel forms across the cell membrane and the virus enters through invagination from which process it enters into the host encapsulated inside a vesicle. It is thus protected from the host's antibodies.
  3. Uncoating - Host cell enzymes (from lysosomes) strip off the virus protein coat (a mistake committed by the host). This releases or renders accessible the virus RNA.
  4. Replication - Viral RNA polymerase copies plus-sense genomic RNA into complementary minus-sense RNA, the whole thing becomes cDNA (complementary DNA). The cDNA is then integrated into the host DNA for replication using the host's replicating machinery.
  5. Synthesis of viral Proteins - For single-stranded, positive-sense RNA virus, the RNA itself is the mRNA, which uses the host's ribosome and tRNA (also aaRS by implication) to make the viral coating proteins and viral RNA polymerase.
  6. Assembly - When sufficient plus-sense progeny RNA and virion proteins have accumulated, self-assembly begins with the virions (newly formed viruses) enclosed inside a vesicle.
  7. Release - The viruses, now being mature are released by either sudden rupture of the cell, or gradual extrusion (budding) of enveloped viruses through the cell membrane. In the case of bacterial viruses, the release of progeny virions takes place by lysis (destruction) of the infected bacterium. However, in the case of animal viruses, release usually occurs without cell lysis.


Some Comments :


The RNA virus equips only with (a) a single strand of RNA as template for replication as well as for translating its codes to viral proteins, (b) the viral polymerase for inducing replication by the host, (c) the protein coating to provide an enclosure. It relies on the host's DNA polymerase for the actual replication of its RNA, and the host's ribosome, tRNA, aaRS, etc. for the translation of its RNA codes to proteins. There are numerous kinds of viruses beside the RNA virus. It seems they belong to a class of "organisms" failing to complete the transition to life. They lay dormant until a favorable condition to wake up. Relic of such death-end products from the prebiotic era could still be around somewhere that we are not aware of. The viruses do manage to generate a system capable of self-assembly (Figure 19). However, they fail to incorporate ATP usage into their system and thus no energy is

Figure 19 Virus Self-assembly [view large image]

available to promote the system to non-equilibium state - a prerequisite for self-organization and onto real living organism.

• The viruses can be considered as the failed experiments in the trial and error processes toward the origin of life.



• Physics and Chemistry - (a) Energy the prerequisite for life, (b) Solvent to promote chemical reaction, (c) Enzyme a necessary substance for metabolism (chemical reaction for complex molecules).
  
  
  The requirements in detail :
  
  
  • (a) Energy -
    
    
    • Carbon Atom - The ability to form covalent bonds with other carbon atoms in long chains and rings distinguishes carbon from all other elements. This property of carbon, and the fact that carbon nearly always forms four bonds to other atoms, accounts for the large number of known compounds. At least 80 percent of the 5 million chemical compounds registered as of the early 1980s contain carbon. The affinity of carbon for the most diverse elements does not differ very greatly - so that even the most diverse derivatives need not vary very much in energy content. This ability allows the organic world to exist in a special form of thermodynamic stability.

      		The electron configuration of the normal carbon atom has 2 electrons in energy level 2S and 2P respectively. By supplying about 2 ev to a carbon atom, the 4 electrons in the 2S and 2P states are rearranged to the SP3 state (Figure 20). The four electrons in the SP3 state form the tetrahedral arrangement (Figure 21) of orbitals (probability distribution of electrons), which can form stable covalent bonds with other atoms.
      Figure 20 SP3 State [view large image]	Figure 21 Tetrahedral Structure

    
    
    • Non-equilibrium State - Creation of a tetrahedral configuration is the basic reason for pumping energy into biological system to maintain metabolism and cellular structure. Therefore, the biological system is said to be in a non-equilibrium state. The electrical discharge in Stenley Miller's experiment is an example of infusion energy to move the molecular configuration into the non-equilibrium state. The continual free energy input from the environment will lead to a dissipative structure (Figure 22), which is a necessary condition for life. The process is called dissipative because it continuously dissipate free energy (work) into high-entropy

      energy namely heat. However, not all dissipative structures are living systems, non-life examples include convection, hurricanes, the Solar system, and galaxies, ... Living system can form only when the dissipative structure begins to perform work. As the hybrid orbitals of the tetrahedral configuration do not exist in an isolated atom, but arise while the carbon atom is interacting with others to form a molecule, it will dissolve and return to an equilibrium (lowest energy) state once the input of free energy ceases causing the removal of the associated constituents.

      Figure 22 Dissipative Process [view large image]

      BTW, life is optimized to an near-equilibrium state to make it a highly efficient process in using free energy.

    
    
    • Entropy - Beside the infusion of energy to support life, the organization of complexity requires the removal of entropy as

      shown in Figure 22. Such requirement is fulfilled by re-transmission of lower frequency radiation back to the cold dark space as illustrated in Figure 23. The process re-emits more photons (at lower frequency) back to the space. By Boltzmann's definition of entropy, it has the effect of enlarging the phase space volume and hence returning more entropy to space than receiving from the sun in accordance with the second law of thermodynamics.

      Figure 23 Entropy and Life [view large image]

      Ultimately, it is the stars which generate the carbon atoms (see "Origin of Elements"), and the Sun which supplies energy to all life on Earth.

    
    
    
  • (b) Solvent -
    
    
    • Property - A solution is a mixture in which one substance called the solute (the component that changes state upon dissolving in smaller amount) is uniformly dispersed in another substance called the solvent. Because the solute and the solvent do not chemically react with each other, they can be mixed in varying proportions (see Figure 24). Solutes and solvents may be solids, liquids, or gases (see Figure 25). Gases form solutions easily because their particles are moving so rapid and far apart so that attractions between particles are not important. When solids or liquids form solutions, there must be an attraction between the solute particles and the solvent particles. Otherwise, the particles do not mix and no solution forms. For example, compounds containing nonpolar

      		molecules such as iodine, oil, or grease do not dissolve in water because water is polar. The polarities of a solute and a solvent must be similar in order to form a solution. It is found as early as the 1800s that whenever a substance is dissolved in a liquid the vapor pressure (the pressure at which the rate of evaporation is equal to the rate of condensation) of the solvent is lowered. Such behaviour has the effect of rising the boiling point and lowering the freezing point of the solution. It also produces osmosis, which is the flow of a solvent,
      Figure 24 Solution	Figure 25 Types of Solution [view large image]	usually water, through a semipermeable membrane into a solution of higher solute concentration, i.e., diluting the solution.

    
    
    • Water - In the H₂O molecule, the electric field around the oxygen atom is stronger than that around the hydrogen. The electrons from the hydrogen atoms are drawn close to the oxygen. This leaves the hydrogen atoms positively charged at one end as shown in Figure 26. Hydrogen bond is different from the covalent bond since there is no orbital overlap; it is not an ionic bond since there is

      		no charge transfer from one atom to another. The strength of the hydrogen bond is about 10 times weaker than the covalent and ionic bonds. It is important in fixing properties such as solubilities, melting points, boiling points, and in determining the form and stability of crystalline structures. Molecules such as water carrying hydrogen bonds are called polar molecules. They play a crucial role in biological systems.
      Figure 26 Hydrogen Bond	Figure 27 Water [view large image]

      Water has some special properties crucial to the existence of life courtesy to the hydrogen bond (Figure 27) :
      
      
      1. Heat Capacity - Water has high heat capacity (needs more heat to rise the temperature) and boiling point by virtue of the additional hydrogen bonds that keep the water molecules packed together with extra strength (see Figure 27a, white bar represents covalent bond, grey and blue bars for hydrogen bond). This property moderates the temperature of the environment. Lack of water turns the land into desert. The same hydrogen bonds also pull water up a tree, in a phenomenon called capillary action, and maintain surface tension, which enables small bugs to walk on water.
      2. Ice - When water cools to below 4^oC, it suddenly becomes less dense. It is because the thermal agitation cannot overcome the hydrogen bonds and solid ice begins to form. The angle subtended by the two hydrogen atoms then increase from 105 to 109 degrees resulting a less tightly packed configuration (Figure 27b). That's why ice can float on water and protects the water beneath from frozen allowing fish to live in the polar regions or creatures under the icy surface of Europa (Figure 27f).
      3. Solution - Water is a good solvent able to dissolve many polar substances (Figure 27c). That's why water-based liquids, like blood, are perfect transporters of essential substances such as salt, sugars and hormones.
      4. Protein folding - Some amino acids are hydrophobic (repelled by water); while others are hydrophilic (attracted to water). In a a liquid environment such as the inside of cells, these properties dictate how a protein "assembles", or folds (Figure 27d). DNA too, depends on water. Experiments have shown that the double helix would fall to pieces without water. This is because water molecules help form hydrogen bonds between DNA's phosphate groups.
      5. DNA binding site - Some biochemists think that water molecules may play an active role in guiding enzymes to certain spots on the DNA (Figure 27e). It is found that water molecules spend more time around certain areas of DNA when cell divides. Such tendency seems to suggest that water is needed for the expression of genes - a process that is kick-started by binding the transcription factor to the gene switch.
      6. Origin of Water on Earth - Water molecules are one of the components in interstellar dust. It would be incorporated into the primordial Earth naturally. Water as many other gaseous materials are trapped by the Earth's gravity when it acquired a sufficient amount of mass. It might be in the form of solid when the temperature was too low in the very beginning, and in the form of gas when it was covered with magma produced by planetesimal impacts. Only in a very narrow window of temperature (from 0^oC to 100^oC) it turned into liquid state suitable for life. Later on more water are added via the impacts of comets, which contain a lot of water and the impacts occurred more often in the past.
      7. Search for ET - All known life on Earth depends on water. Typical enzymes, just like DNA, need water to function properly. Nobody know if enzymes can function in another liquid, or there might be fundamentally different life-forms in other parts of the universe - a possibility that would not be ruled out by many scientists. Nevertheless, NASA has chosen the strategy of "Follow the Water" in its programs to search for extra-terrestrial life (to Europa for example, see Figure 27f).
    
    
    • Internal Sea - Some theories on the "Origin of Life" suggest that life arose from the sea. Specific environment varies according to different scenario. It could be in hydrothermal mounds or mud pool or tidal pool. While composition of the ancient sea 4 billion years ago could be different from the present; very recent sample shows that it is not much different from the interstitial fluid bathing all cells in multicellular organisms, i.e., it is salty with lot of Na⁺ and Cl^- ions (Figure 28). The importance of interstitial fluid was observed back to 1887 by Claude Bernard. Its role has been reinforced in modern time by such similarity.

      While unicelluar organisms can acquire nutrients and expel waste directly in aquatic environment, they could not survive on dry surfaces with a humidity of less than 10%. The same situation is applicable to the cells in multicelluar organisms which solve the problem with the "Internal Sea" in the form of Interstitial Fluid (ISF). Figure 28 also shows that the fluid composition inside the cell (Intracellular Fluid, ICF) is quite different from sea water as the constituents are altered by the metabolic process of life.

      Figure 28 Sea Water and Body Fluids, Composition [view large image]

      See "Interstitial Fluid" for more detail.

    
    
    
  • (c) Enzyme - The rates in a chemical reaction is measured by the amount of reactant used up, or the amount of product formed, in a certain period of time. Reactions with low activation energy E_a go faster than reactions with high activation energies. The rate of a reaction can be affected by temperature T, the amounts of reactants in the container (concentration [A]) as shown by the formulas in Figure 29, pressure and cross-section of the reactants have similar effect of concentration by varying the chance of collisions; while for large macromolecules, catalysts is required to match the reaction sites and to lower the activation energy as shown in Figure 30. Reactions almost always go faster at higher temperatures, because the increase in kinetic energy makes the reactants move faster and collide more often. The rate of a reaction also increases when reactants are added (resulting in higher concentration) since there are more collisions

    		between the reactants. Another way to speed up a reaction is to lower the activation energy Ea. This can be done by adding a catalyst, which speeds up the reaction but is not itself changed or used up. Enzymes are known to catalyze more than 5,000 biochemical reaction types. Most are proteins, although a few are RNA molecules (ribozymes). The catalyzing function comes from the unique three-dimensional structures.
    Figure 29 Reaction Rate for Simple Molecules [view large image]	Figure 30 Enzyme [view large image]	Enzymes are classified according to the chemical reactions they catalyze. See "Enzyme Commission (EC) number".

    
    Two opposed trends on the formation of an enzyme : on one hand it needs to fold into a stable and compact structures, while on the other hand they must also be flexible in order to catalyze chemical reactions. The active site of an enzyme is highly strained because it is designed to develop favorable interactions with the transition state of the catalyzed reactions. This strain diminishes the stability of the global structure of the enzyme and thus a trade-off between stability and function has to be established. It is found in the lab that enzymes exhibit a remarkable evolutionary adaptability. Many enzymes evolved from pre-existing enzymes via gene duplication thus allows the evolution (of enzyme) from a simple version to more complication one via the process of Darwinian selection (see "A Darwinian Process: The Molecular Evolution of Enzymes").

  A Scenario of Prebiotic Evolution : based on the sequence of events and related information cited above, a scenario is constructed below for the evolution of inanimate matter in a prebiotic world toward life (see illustration in Figure 31).

                             ( The Beginning,  Enzyme Catalysis,  Uncorrelated Evolution ,  Self-assembly ,  Pre-requisites for Life )
  
  
  1. The Beginning -
     • In the beginning energy provided by the Sun had made the Earth warm enough to have H₂O molecules in liquid phase forming the oceans, rivers, lakes and ponds.
     • The atmospheric composition at that time had N₂, H₂O (water vapor), CO₂, and some CH₄, NH₃.
     • In a rather similar environment, the Stanley Miller's experiment is able to make : carboxylic acids (R-COOH), nucleic acids base, and amino acids. Since the experimental environment is not exactly the same as the atmospheric composition of the early Earth (note the presence of CO₂, and the absence of H₂), it could be that those organic molecules were produced in localized spots where the chemical composition may be different from the global environment. Another possibility is the delivery by meteorites. Regradless of the question of its original source on Earth, astronomical observations have detected lot of those simple organic molecules in space. They had to be around in the environment of early Earth.
     • The amino acids can be synthesized to oligopeptides (short peptide chain folding to form protein) by removing the water molecule between the link. However, it can also became hydrolyzed (breakdown) by adding water to the product. Thus the more complicated polymers are easier to make in mud pools or tidal pools where the wetting and drying cycles permit easier synthesis and also allow the necessary degrading back to monomers for making new products.

     Polymerization of oligonucleotides (short polymer of nucleotides in the form of RNA) joins together the nucliotides, similarly to other biological molecules, by condensation reaction that releases a small molecule. Unlike proteins, carbohydrates, and lipids, however, the molecule that is released is not water but pyrophosphate (O3-P-O-P-O3 = ppi). This is essentially the ATP hydrolysis to AMP reaction with the release of a lot more free energy resulting in higher binding energy. That's why polymer of nucliotides, e.g., RNA or DNA does not break down easily.

     Figure 31 Prebiotic World [view large image]

     All such short polymers can be produced at moderate temperature range and sufficient concentration without the assistant of enzyme (Figure 29).

     
     
  2. Enzyme Catalysis -
     • As the molecules become larger and the configuration more complicated, reaction between two of them requires more precise contacting point (active site). It needs an enzyme to arrange them in proper orientation for the reaction to proceed.
     • The oligopeptides or oligonucleotides would coil into a three dimensional configuration from the linear chain by hydrogen bonds. The product is somewhat malleable and can accommodate two particular molecules for a reaction to take place. This is called catalyzation without which there would be no macromolecules and no life.
     • Beside the lock-and-key specification by the enzyme, some enzymes also supplies energy in the form of ATP (AMP + energy) to establish the C-C, C-N, C-O, or C-S bonds (see Figure 17 and Figure 31,2).
     • Other classes of catalysis do not use ATP, those enzymes could be the earlier version of the more complicated one. For example, the hydrolases are the enzymes in hydrolysis (breakdown) of many polymers without the involvement of ATP. In fact, enzymes in Enzyme Classes EC-1 to EC-5 do not use ATP, the one class that does belongs to EC-6.
     • The catalysis can either join two molecules together or breakdown a molecule. The appearance of such mechanism on the scene accelerated all kinds of reactions by ten- to a hundred-million-fold permitting lot of trial and error processes to run - often without any consequence, i.e., coming to a death end.
     
     
  3. Uncorrelated Evolution -
     • The system is very slowly evolved to a collection of increasingly complicated molecules, everyone of them acting alone without regard to others. The lack of design is compensated by the sheer number of trials, very small incremental change in atomic scale, and plenty of time in the order of billion years. The tidal pools could serve as enclosures to retain the changes which would be lost in the vast ocean otherwise. Among the multitude proteins in such environment, two enzymers stand out for their role in the evolution toward life. They are the proto-polymerase and proto-synthetase (proto = primitive). There are also specialized forms of RNA such as the mini-helix and proto-mRNA.
     • Mini-helix is single RNA strand folds back to form double helix, which becomes part of the proto-tRNA.
     • Proto-mRNA is the precursor of modern mRNA, which has a AUG start codon at the 5' end and a UGA codon at the 3' end together with the untranslated regions (UTRs) before the AUG and after the UGA.
     • Modern polymerase is an enzyme that synthesizes long chains or polymers of nucleic acids. It is used to assemble DNA or RNA molecules, by copying a DNA or RNA template strand using base-pairing interactions. There are different types of

       	polymerase; for example, the RNA polymerase is composed of a dozen different proteins. Together, they form a machine that surrounds a DNA strands, unwinds them, and builds an RNA strand based on the information held inside the DNA. Once the enzyme gets started, RNA polymerase marches along the DNA copying RNA strands thousands of nucleotides long (Figure 32).
       Figure 32 RNA Polymerase in Action [view large image]	Another example is the DNA replication expressed by the formula : deoxynucleoside triphosphate + DNAn + DNA-pol diphosphate + DNAn+1 + DNA-pol

     • Modern synthetase is another enzyme that catalyzes the linking together of two molecules usually using the energy derived from ATP. The most cited example is the aminoacyl-tRNA synthetase (aaRS) as shown in Figure 17 in the process of translating the genetic codes to peptide chain. The process can be expressed by the formula : Amino Acid + tRNA + ATP + aaRS Aminoacyl-tRNA + AMP + PPi + aaRS.
     • The proto-polymerases and proto-synthetases did not perform the functions executed by their modern cousins. In fact, all the macromolecules did not have permanent association to each other - their evolutions are uncorrelated (Figure 31,3). This step is illustrated by Phases 1 and 2 in Figure 14. Expansion of these primitive RNA and peptide was achieved by appending basic unit or short oligomer one at a time to the linear sequence. The 3-D structure is produced by folding.
     • It is the interplay between activation energy and concentration that bumps up complexity of molecules. Lower activation energy for certain reactions would increase the concentration of such end product, while higher concentration of that species would overcome a little bit the activation energy for another reaction to produce a new kind, and so on, ... with the enzyme along the way to facilitate

       the process by lowering the activation energy. (see Figures 29 and 30 respectively for the relationship between reaction rate and concentration and the effect of enzyme in lowering the activation energy). The absence of reproduction to accumulate the same kind is covered by stability, which is in turn related to stronger binding energy (Figure 33). Thus, such chemical species would be preserved. Anyway, only perishable item needs to be replaced or reproduce in order to keep its presence.

       Figure 33 Binding Energy [view large image]

       For example, the binding energy of water H2O is stronger than either H2 or O2 (see Figure 33). It sticks around on Earth for 4 billion years without any mean of reproduction.

       BTW, the hydrogen bond in most macro-molecules is about 10 times weaker than the covalent bond in H₂O (see Figure 26). It is found that the secondary structure of macromolecules can be affected by external conditions, including pH, temperature, and ionic interaction beside the binding energy in an isolated environment. Proper native secondary structure is an important aspect of the development of stable formulations. See a comparison of stability between DNA, RNA, and Protein.
     
     
  4. Self-assembly -

     A space enclosed by a wall is a prerequisite to assemble complex chemicals. It would keep those macromolecules inside but allow other molecules to pass through. Surfactant molecules (such as the lipids) contain both hydrophobic groups (the tails) and hydrophilic groups (the heads). They form double layers spherical vesicle naturally with the hydrophilic heads facing the aqueous solutions on the outside and the hydrophobic tail pointing to the space between the layers (Figure 34). The modern cell membrane is a form of such vesicle.

     Figure 34 Vesicle [view large image]

     The capsid of the virus is a protein shell in various forms as shown in Figure 19. This special coating is made for penetrating the membrane of the host. It may also contain a lipid envelope. The

     minimum linear size for modern cells is about 10^-5 cm, viruses can be 10 times smaller with less biochemical machinery inside.

     	In a nutshell, self-assembly just follows the law of physics, which tends to drive a system to an equilibrium state with minimum energy. It is as natural as a rock rolling down from hilltop to the bottom of the valley where the gravitational potential Ep is at its minimum (Figure 35). Such state would be easier to achieve in a small volume than globally with a lot of things to be adjusted.
     Figure 35 Minimum Energy [view large image]

     • A good example of self-assembly is the operation of ribosome in modern cells. It collects all the components such as the LSU, SSU, tRNA, and a host of assorted proteins together in the presence of mRNA (see "Modern Day Ribosome"). After the assembly, the aggregate switches to self-organization to run the translation. It turns into a state of non-equilibrium - the trademark for life.
     • As mentioned earlier about virus assembly, it lacks some important components to replicate itself. The problem can be attributed to mistake in self-assembly, i.e., it fails to include all the necessary apparatuses into the enclosed volume. In the game of trial and error, somehow some self-assemblies (in small tidal pools for example) did incorporate everything correctly for use in the future. Such assembly could become the precursor for a living entity (see Figure 31,4). This step would be represented by Phase 3 in Figure 14. As shown in the same figure, there is a few more steps (Phases 4. 5, 6) before reaching the "Origin of Life" symbolized by LUCA. By then the system will be running in non-equilibrium state and its behavior will be dictated by the rule of self-organization.
     
     
  5. Pre-requisites for Life : There are some components that have to couple and evolve together before life can start to run on its own.

     RNA - For the positive sense RNA, it can also serve as mRNA to encode the genetic code for translation. Initially, it would be just a collection of randomly arranged nucleotides. It has to be optimized to become gene for making protein and other functions (Figure 36). RNA Polymerase - It uses the reverse transcriptase (RT) enzyme to create a double stranded cDNA, which is a copy of the RNA itself matched by a complementary strand to form a DNA double helix (Figure 36). In virus, replication of the viral RNA is make from its cDNA by hijacking the host DNA polymerase. In modern laboratory, the process is used as one of the methods in polymerase chain reaction (PCR), and also in molecular cloning. In the prebiotic era, the cDNA is very likely to be the intermediary in the transformation from RNA to DNA as the genetic codes keeper.

     Figure 36 Pre-requisites for Life [view large image]

     tRNA - It serves the important function in the translation of genetic codes to amino acids. The bending and looping structure seems to indicate its ancient root as "ribonucleo-protein".

     • aminoacyl-tRNA Synthetase (aaRS) - This is the enzyme to charge the tRNA by linking it up with an amino acid (corresponding to the tRNA's anti-codon). The translation would not work without it. It started to evolve from ancient past as described by the theory of "Protein/RNA World".
     • Ribosome - Every living organism has a version of this organelle running all the time. At the presence of mRNA, the ribosome self-assembles many parts into a whole to make protein according to the genetic codes in the mRNA. It's evolution has been described at length in the "Top-down Theory".
     • Membrane - The enclosure needs pores to drain out wastes such as CO₂ and to admit nutrients such as small monomers and ions. It should also have the capacity to expand and to divide when there is too much material inside - a trait for cell division.
     
     When all these components have self-assembled and self-organized, the entity starts to live. The imperfection and imprecision will be resolved according to the rule of "Natural Selection" - that's another story.
     
     
     

     	Footnote : ATP Hydrolysis ATP (Adenosine TriPhosphate) is the adenosine nucleotide with 3 phosphate groups. Hydrolyzing each phosphate would release an energy of ~ 0.3 ev, i.e., ATP + H2O ADP + Pi + 0.3 ev, or ATP + H2O AMP + PPi + 0.6 ev. The A, G, C, T (or U) nucleotides in DNA or RNA retain only one phosphate group as the backbone to link up the structure (Figure 08b).
     Figure 08b ATP, ADP, AMP, and RNA Backbone [view large image]	ADP = Adenosine DiPhosphate AMP = Adenosine MonPhosphate

     
     

     		Sun light is captured by chloroplasts to make glucose, which in turn is used by the mitochondria to produce ATP (Figure 08c). The ATP Synthase is ultimately responsible for the chemical process. The chemical formula on the (peptide)n to (peptide)n+1 reaction (and the ATP to ADP, AMP formulas above) actually require a H+ ion on the right-hand-side to make them balanced. It is the concentration gradient of such ions across the cell membrane to complete the conversion from ADP to ATP. Figure 08d is a cartoon to show the role of ATPs in modern life.
     Figure 08c Eco-cycle	Figure 08d Food Cycle [view large image]