CHAPTER-1

The year 1859 is marked with the publishing of Origin of Species, by Charles

Darwi~. Perhaps the thesis put forth in this work was, and is, most c~gent statement

on the evolution of life on Planet Earth. The Theory effectively gave birth to the new

scientific principle and emphasized the central theme that the life evolves under

selection pressure and has a direction. The footprints of selection pressure can be

observed and analysed by studying the variation in morphological traits of the species.

The pervasive affect of the theory of evolution permeated natural and social sciences.

Subsequent development not only reinforced the Darwinian evolutionary concepts but

also shifted the focus of attention on the problem of "Origin of Life". The realization

that life is sustained as a special case on Planet Earth and it only occupies a few metres

thick surface provided impetus for the emergence of the attempts to search for the

answers in chemical basis of Origin of Life. The chemical basis undoubtedly were

closely linl,ed with the environment- past and the present( Oparin, 1953 ). Different

theories addressed different aspects linked to the origin of life to provide scientific

reasoning at different stages. The research associated with the problem of Origin of Life

holds universal importance for the scientists and non-scientists. Undoubtedly, most

persuasive reason to pursue research in the area of origin of life comes from the human

quest to seek answers about its own origin, which holds eternal intrinsic fascination.

The existing understanding of theory of Origin of Life, over last five decades,

have undergone a rapid change in providing scientific explanation and a common

acceptable understanding has emerged about the sequence of events which has

2

resulted in the emergence of life on Earth. This broad understanding still suffers from

serious lack of explanation and plausible scientific theory, which can interpret the

growth of information contents of information bearing biomolecules, Deoxyribose

Nucleic Acids (DNA) and RiboNucleic Acids (RNA); that too in the absence of the

enzymes during pre-biotic molecular evolution. This aspect is often ignored while '

presenting the broad scientific scheme to explain prebiotic aspects of Origin of Life.

Most of the steps in this sequence are derived from experimental evidence, done over

last six decades. Origin of life problem deals with two distinct phases; i) pre-biotic

molecular evolution leading to the appearance of cell and ii) evolution of life per se.

The work presented in this thesis pertains to the formulation of a theory as a part of

pre-biotic molecular evolution, which provides the thermodynamic basis of

information growth in enzyme free environment. Consequently, the present status of

pre-biotic molecular evolution and its shortcomings are presented in detail.

Pre-biotic Molecular Evolution

The concept of pre-biotic evolution emerged from the " germ theory of disease",

which emphasized that the spontaneous appearance of life does not occur and life

could only have emerged from non-living in the past, in a slow process of evolution.

Schrodinger ( l 944) raised some pertinent questions and these queries provided the

foundation for the emergence of the chemical basis of molecular evolution. The study

of pre-biotic evolution broadly can be divided into three stages;

3

EARTH'S OR1Grn

Permissible' Ring ofLife

Figure 1

a) geophysical aspects, b) chemical aspects and c) biological aspects. The

theories formulated had to take into account the geophysical aspects as, the

environmental conditions define the fundamental constraints on which the pre-biotic

molecular evolution must have occurred. The present day understanding of the origin

of life, in a way, has taken the scientifically accepted form.':fhis form has emerged

and evolved over a long period, where contribution by some scientists who have

addressed different aspects of the Origin of Life stands out(Darwin, I 858; Haelde,

1834-I919; Oparin, I924; Miller, I952; Fox,I954; Orgel,I973 ; Cech,1989

Spegelberg, 1967 ; Eigen and Schuster, I 978; etc).

4

Pre-biotic earth's atmosphere

The formation of the planet earth and its location in the solar system is

often thought to be a part of the fundamental condition for the evolution of life.

Figure 1 shows the approximate band at a distance from the sun, where it will be

possible for the life to evolve. The average distance of earth from sun is 1.496 x 108

km (Miller and Orgel, 197 4 ). Earth is one of the 288 planets where life could

evolve, as sho~ in figure 1 (Martini, 1998). The Present estimates of the age of the

earth range from 4.5 to 4.7 aeonst ( Salthe, 1972, Orgel and Miller, 197 4 ). The

oldest rocks are dated 3.3 to 3.6 aeons, and since these rocks are partly

metamorphic, they must be having a solid crust (lithosphere) sometimes prior to

this. The first sedimentary rocks seem to be 2. 7 to 3.2 aeons old which provides

the evidence for the weathering of the rocks exposed to the primitive earth=s

atmosphere, by that time. The earliest rocks also show evidence of having been

solidified under water, which suggests that the hydrosphere on earth antedates the

lithosphere. In this period, when the first sediments were being deposited under

water, it is felt that the original atmosphere of the earth received significant

amount of methane (CH4), ammonia (NH4) and hydrogen (H2) from these

deposition (Salthe, 1972 ). These gaseous constituents, once in atmosphere, broke

down into their elemental form. Subsequently they (CH4 , NH4 and H 2 ) were further

released into the atmosphere in the form of volcanic activity from the interior of the

earth. This secondary volcanic emissions also contained other major constituents

t 1 aeon = 109 years

5

like; water vapour (H20 ), carbon dioxide ( C02 ) , carbon monoxide ( CO), nitrogen (N2) ,

sulphur dioxide (S02 ) and hydrochloric acid (HCl). The schematic diagram of earth's

primitive atmosphere is shown in figure 2 (Martini, 1998). The sea during this

NH3

. LavaFlow

Figure 2. Primordial Atmosphere Rich in Gasses Like NH 3 , SO 2 and 0 2

period is considered to be having comparable salinity with the today's sea

(Brancazio et. al,l963). The chemistry of the sea favoured precipitation of

carbonates from the water. The remaining NH3 , after its initial loss from the

atmosphere, was dissolved in the sea. Under these conditions, it is easy to conclude

that the silicate buffering system must have maintained the sea at an alkaline pH.

The alkanity of sea water provided the essential boundary condition within which the

chemistty of life could operate.

The oldest known fossils , blue green algal stromatolites, are present in the

6

sediments (dated at 3.2 aeons) found in South Africa. This discovery further narrows

down the approximate time period for the origin of life on earth. The fundamental

steps leading to the evolution of life on earth, in other words, must have emerged

between 3.6 aeons( formation of hydrosphere) to 3.2 aeons (discovery of oldest fossil

records) before present (BP) . The common consensus among scientists concerning the

conditions prevailing on the earth, during these times, are given in table 1. Details

of the geological time scale of earth and the corresponding events related to the

evolution of the life are shown in Table 2( Moore, 1993).

TABLE I F - f b" d eatures o pre IOtlc an present eart h' s sea an d h atmospJ ere Prebiotic sea Present sea Pre biotic Present

atmosphere atmosphere

Moderately salinity 35 % saline (Davis, 1987b) Reducing Oxidising

(Salthe,1987)

Shallow alkaline pH 7.8 CH4,NH3 ,H2 ,H20 , N 2 , 0 2 , Ar, C02 ,

(Salthe, 1987) (Davis, 1987a) C02 , CO, N2 , S02 , Ne, He, CH4 ,

HCl Kr, N20,H2 , Xe

In contrast to todays atmosphere, which is oxidizing, the prebiotic earths

atmosphere was reducing. The oxygen was only present predominantly as a part of

H20. Oxygen, however, could be freed from water by photolytic splitting of water, but on

its release, it immediately combined with carbon, silicon or sulphur to form CO, C02

,

Si0 2 and S02 . Essentially, the reducing atmosphere ensured the anaerobic·

conditions during these time.

7

Table 2 :

Phanero7;oic

Proterozoic

Archean Hade. an

Triassic

Carboniferous

Glaciation in northern hemisphere .. Human beings worldwide. Extinction of large mammals. Continued diversification of modern birds,angiosperms, and placental mammals. By the end of the Tertiary the genus Homo bad appeared. Gra.sses and grazing mammals become abundant.

Beginning of radiations of flowering plants. Dinosuars abundant then extinct Many other extinctions possibly associated with of meteor at the end of period. First known birds and mammals. Dinosaurs abundant. First dinosours. Cycads and conifers abundant. Continents moving apart Land masses form single continent, Pangea Frigid conditions and massive extinctions at end of period. Mammal like reptiles. Warm humid condition result in huge forests of primitive plants, which formed extensive coal deposits. Reptiles appear at end of period. Insects presents Trilobites, brachiopods and crinoids abundant. Labyrlnthodonts and jawed fishes abundant. Age of fishes. First amphibians. Land plants · and land arthopods abundant. Atmospheric oxygen at present levels or highei'. Continents moving towards one another. liracbiopod·s~ . echinoderms and cephalopod~. ·abundant. First insects·. Brachiopods, trilobites and eurypterldes common. First jawed fishes. Plants and animals invade land. Atmospheric o.xygen about lO%. Earliest known vertebrates. Bracb;iopods, trilobites, cephalopods and graptolites common. Probably all metazoan phyla preseQt. Trilobi~ and brachiopods abundant. Atmospheric · o~.yge.n reaches 1%. Oldes:t.known metazoans. Coelenter-tes, ilnnelidst and arthropods may have have been present,

· Abundant prokaryotic life. Eukaryotes may have appeared by 2~000 million years ago. Atmospheric oxyg¢n about 0.2%. Oldesf Known rocks and prokaryotes. Earth forms . No geolOgical record.

In the absence of free 0 2 the upper atmosphere (Stratosphere) was devoid of

any ozone (03) shield, which allowed the peneteration of harsh ultraviolet (UV)

radiation to the ground (Fox 1964). The presence of harsh UV radiation ensured that

early steps leading to the evolution of life could only evolve under water, water

could effectively filter out the high energy UV. In fact, a strong correlation exists

between the increase in the diversity of life on earth and the formation of 0 3 layer,

shown in figure 3.

Scientific evidence suggests that 3.2 aeons ago , lime-secreting (CaC03 )

blue green algae were already present. Further interesting observation is that the

dome shaped calcareous sedimentary structure , stromatolites, organised around the

blue green algae which suggests that they were inter-tidal-organisms . This indicates

that the moon was present at that time. The scientific conjectures concerning the

composition of pre-biotic atmosphere of earth defines the framework for the abiotic

material chemistry (Sal the, 1971 ).

All theories, which explain the subsequent evolution of life on earth, after

Origin of Life per se, inherits the broad aspects of paradigm conceived by Lamarck;

Chales Darwin and Mendel. However, the sequence of origin of life form

abiotic =}biotic==}Cell

still holds inquisitiveness, and the problem in itself retain intense fascination. In this

regard the various aspects related to pre-biotic chemistry involving

9

Figure 3 . __ . _ . The relationship between changes in atmospheric oxygen levels and some of the major stages that arc believed to have oocured during the evohrtion of living organisms on earth. As indicated, geological evidence suggests that there was more than a billion -year delay between the rise of cyanobacteria ( thought to be the first organism to release O:\')'get!) and tbe·timc that high oxygen levels began to accumulate in the atmosphere. This delay was due largely to the rich supply of di'>SOivcd ferrous ion in the oceans, which reacted with the released oxygen to form enormous iron oxide deposits.

20

O.XVGEN lEVELS IN

ATMOSPHERE 10 ( .,. )

• TIM.E

(BILLIONS OF YEARS) 0 I I

I tl

stort of rapid 02 accumulation (Fe2•in octZans UStld up)

2 -·-1 .,. 4 . L---.---1 prtZstZnt day

3

I lformation of oceans and continents first first wattZr splitting origin of tZucaryotic first vtZrttZbrattZS

formation of the earth

living photosynthesis cells releases 02

first photosynthetic

CtlltS

ctZIIs(algae)

atZrobic respiration

btZcomes widespread

first multicellular plants and anirnc:als

L-------------------------·--,----...1

abiotic =} biotic transformation is summarised below.

Pre-biotic Chemistry

The landmark experiment done by Urey and Miller (1952) proved to be a

clinching experiment which could show that under simulated pre-biotic conditions it

was possible to expect the formation of large amount of biotic material from abiotic

material. This experiment was in the the bael<ground of the early work done by Haclde

(1858). This conception, subsequently, was explained on scientific principles by

Oparin in 1 924. The idea of spontaneous formation of biologically significant

molecules from inorganic precursors was independently coined also by Haclde (1858)

and Oparin(l924). The theory propounded by Oparin set the stage for the emergence

of theoretical concepts to explain the chemical evolution of life, starting from pre­

biotic evolution.

Orthodox Chemical Evolutionary Theory

The theory developed by Oparin during l 920-l930s is known as "chemical

evolutionary theory". Oparin had a much more accurate understanding of the

complexity of cellular metabolism, but during his time he could not fully appreciate the

complexity of the molecules such as protein and DNA that mal<.e life possible. Oparin,

like his nineteenth century predecessors, suggested that life could have first evolved as

11

a result of a series of chemical reactions'?. He, however was able to envision that the

process of chemical evolution would involve many chemical transformations and

reactions would have spread well over many hundreds of millions (or even billions) of

years (Meyer, 1998).

Oparin also believed that the gases; such as ammonia (NH3), methane (CH4),

water (H20), carbon dioxide (C02 ) and hydrogen (H2); would have rained down into

the early oceans and combined with metallic compounds extruded from the core of the

earth. In the presence of UV radiation from the sun, the ensuing reactions could have

lead to the formation of energy-rich hydrocarbon compounds like ATP.

These in tum would have combined and recombined with various other compounds

to mal<:.e amino acids, sugars, phosphates and other "building blocks" of the complex

bio-molecules (such as proteins), vital to living cell. These constituents would

eventually arrange themselves into a simple cell-like enclosures that Oparin called

coacervates. Oparin then proposed a kind of Darwinian competition for the survival

of coacervates. The survived coacervates could sustain increasingly more and more

complex molecules and metabolic processes. The shadow of Darwin's theory loomed

large on the steps conceived by Oparin to account for the origin of the complexity from

initial simplicity. In simple terms Oparin's contribution showed that matter interacting

chemically with other matter, if given enough time and the right conditions, could lead

to the emergence of life. Complex cells could be built from simple chemical precursors

* The details concerning the actual chemistry leading to the emergence of cell did not had any experimental support.

12

without any guidance from supernatural agency. To ascertain these claims, made by

Oparin ( 1924), it took thirty years. Experiments broadly were able to agree with the

concepts of Oprain's theory (Fox and Dose, 1927; Kenton and Steinman, 1969; Miller

and Orgel, 197 4).

The Miller-Urry Experiment

Miller and Urey's experiment probably was the first experiment which attempted

to answer the possibility of the formation of small biom:olecules from inorganic

material, in December of 1952. Miller circulated a gaseous mixture of methane (CH4),

ammonia (NH3), water vapour (H20) and hydrogen (H2) through a glass vessel

containing an electrical discharge chamber. Miller sent a high voltage charge of

electricity into the chamber via tungsten filaments in an attempt to simulate

the effects of ultraviolet light on prebiotic atmospheric gases (CH4 , NH3 , H 20 and

H2). After two days, Miller found 2 percent yield of amino adds in the U-shaped water

trap, used to collect reaction products(Miller, 1953 ). While Miller's initial experiment

yielded only three of the twenty amino adds that occur today naturally in proteins,

further experiments performed under similar conditions have produced almost all of

them. The frequency of occurrence of amino adds in proteins is shown in table 3,

during different times. Other experiments have yielded fatty adds and the nucleotide

bases found in DNA and RNA molecules, but not the sugar molecules i.e. deoxyribose

and ribose necessary to build DNA and RNA molecules. The present day textbook

schematic of Miller experiment is outlined in figure 4(Miller, 19~3).

13

Methane (C~)

Amino adds and Famaldehyde (CH <;/:J)

Aggregation

~ (.._ __ Cd_I_s_iz_~_d.-1 a..::.ggreg=--=-at_es _ ____,)

"" . Cc:mcentratim in "Organic Soup11

Fi~re 4 : Miller's Experiment

The experimental findings could not address the essential microscopic steps coupled

to the emergence of self replicating system(RNA, DNA and Proteins) prior to the

origin of cell'. The Oparin's chemical evolutionary theory has in recent years

encountered severallimitations(Cavior, 1981):

1) Geochemists have failed to find evidence of the nitrogen-rich

"prebiotic soup" essential for Oparin's model.

t Biomolecules central to the theory of Origin ofLife (DNA, RNA and Proteins), at that time were known but nothing was known concerning their structural features. For example, the DNA and RNA structure later unequivocally got coupled with the genetic information contents vital to the theories developed later.

14

Table 3 showing molecular fraction of amino acids per 100 molecules of amino acids in different eras (Florkin et. al, 1961 )

Amino acids Nautilus Aturia Iridina Nautilus Eocene (60 Oligocene ( 40 Holocene Modem

million years) million years ) (10,000 years)

Aspartic acid 8.7 9.0 10.1 9.0 I

Threonine 5.6 3.8 1.6 1.5

Serine 24.0 16.7 7.8 10.9

Glutamic acid 15.6 11.9 5.0 5.5

Proline 3.7 4.8 2.0 1.8

Glycine 20.8 23.3 29.8 35.7

Alanine 9.7 15.2 28.2 27.2

Valine 3.1 5.7 4.0 2.2

Isoleucine 3.1 3.3 2.4 1.8

Leucine 5.6 6.2 4.0 1.9

Tyrocene 0 0 0 2.4

Phenylalanine 0 0 0 2.4

Histidine 0 - 0 0

Lysine 0 - 2.3 0

Arginine 0 - 1.9 0

2) The remains of single-celled organisms in the very oldest rocks (fossil

evidence) testify that life emerged faster than the time it will take for

chemical evolution, as suggested by Oparin.

3)- New geological and geochemical evidence suggests that prebiotic

atmospheric conditions were not friendly, to the production of amino

acids and other essential precursor molecules requited to synthesize

macro-bimolecules.

15

4) In last four decades the developments in the field of molecular

biology has revealed that complexity and specificity of the design of

cells and cellular components, even the "simplest" of cell, defy

simplistic explanation put forth in Oparin's theory (Meyer, 1998).

In view of the experiments and prevalent theories and conceptions it became clear that

the problem posed by the complexity of the cell and its components should be tal<.en

into account. In addition the the new evidence also questioned the probable

composition of the early pre-biotic earth. The new evidence suggested that the

atmosphere was more reducing in its chemical nature. To re-evaluate the Miller-type

simulation experiments under these conditions Stanley Miller (1953 ) conducted

more experiments. In these experiments the the earth's simulated atmosphere

composed of a mixture of reducing gases such as methane (CH4), ammonia (NH3 ) and

hydrogen (H2 ). Earth's atmosphere also contained virtually no free oxygen. Under

these conditions it was shown that basic amino acids could be synthesized. Further

research, done in geochemistry, altered the earlier composition of pre-biotic earth's

atmosphere (Kerr, 1980 ).

The evidence strongly suggested that the neutral gases such as carbon dioxide,

nitrogen and water vapours were present. Ammonia and hydrogen predominated in the

early earth's atmosphere; however no methane was present (Kerr, 1980). In addition,

significant amounts of free oxygen were also present, even before the advent of plant

life. May be, this was the result of volcanic outgassing and the photodissociation of

16

water vapour. This step, leading to the formation of oxygen, appearance of plant and

formation of ozone layer are shown in figure 3.

Miller's experiment was significant for the production of amino acids from

presumably plausible prebiotic conditions. The only reason to continue assuming the

existence of a chemically reducing prebiotic atmosphere is that that the chemical

evolutionary theory seems to require it.

Prebiotic bio-molecules and bio-polymers

Number of experiments simulating the prebiotic origin of biomolecules from

the basic elements C, H, 0 and N show amino acids (or precursors that are

readily hydrolysed to amino acids) are formed with significant yields( Fox and

dose, 1977 ). In proportion, the yield of amino acids and their precursurs were present

in very large amount when compared to the yield of other organic

molecules(Carbohydrates, Hydrocarbons, Nucleotides etc.). Amino-acids; Glycine,

alanine, glutamic acid and aspartic acids are most abundant prebiotic amino -acids

formed( both 1 and d types). It was important to note that the amino-acids formed

were very fit to survive the stresses of various harsh geochemical environments. Their

geochemical fitness is found to be related to; (i) Zwitter-ion character, (ii) high

melting (decomposition) point, (iii) low vapour pressure, (iv) high solubility and

stability in water, (v) selective binding to minerals with cation and anion exchange

capacjty and (vi) ability to undergo intermolecular condensation reactions to yield

17

relatively stable polymers such as polypeptides, proteins and other polyamino adds (

Dose, 1984 ).

Further experiments show that it is possible to form small polypeptides from

simple amino-acids( Fox et.al, 1959 ). Formation of polyamino acid was achieved in

the poly-condensation of free amino acids or dipeptides by simple heating. However,

according to the early literature, glycine and aspartic acids were converted into horny

or colloidal products ( Matsano, 1984 ).

Protenoids

Laboratory experiments, in particular, done by Fox and his associates (1956)

further established that the mixtures of amino-acids can be polymerized under

simulated geological conditions into a variety of individual, non random polymers.

These polymers are similar to the biologically produced proteins having biologically

significant properties. They are therefore often referred to as protenoids. They have

significance in being considered as precursors of proteins, and hence also known as

proto-proteins. Because of their geochemical origins, protenoids or proto-proteins are

considered to be geologically fit molecules(Dose, 1984 ). Cooling of saturated

protenoids in aqueous solutions forms spherical vescicles,named 11spherules" by Fox et

al( 1959 ). Later, this spherical, cell like structures have been called microsperes.

Protenoids rich in acidic amino -acids (aspartic acid, glutamic acid) are generally good

starting material for microspheres production. To some degree protenoids microspere

18

exhibit structures sim1lar to the "organised elements" found in carbonaceous chondrites

or in ancient sediments.

The experiments done by Fox and his group clearly indicated the similarity between

the first protocells of prebiotic evolution 'With microsphere self-assembly formed from

protenoids alone or from the combination of protenoids, lipids and related compounds

which are found also in the membranes of contemporary cells

(Fox et, al, 1959 ).

Proto-cell

The phenomena of microspheres(spherules) was seen as an important step

towards understanding the origin of life (Fox and Dose, 1977). Their properties like;

(i) stabality, (ii) selective permeability 'With osmotic and conductance was similar to

that found in cell membrane, (iii) catalytic activities, (iv)motility, (v)

compartmentalization, (vi) communication between spherules via transfer of material

through morphological junctions, (vii) proliferation via fission and budding and (viii)

growth by accretion. These micro-spheres were accepted as model protocells (Rohlfing,

I 9 84 ) . At this stage, the central issue concerning the emergance of the first self­

replicating cellular machinary was actively persued. Different groups evolved different

theories and theories; in-part, were supported by the experimental evidence.

Origin of genetic code and protein biosynthesis

There are two main scientific views about the origin of the genetic code. First,

view considers that the proteins were the first to appear and they played the role of

19

information bearing bio-polymer. Second, view considers that the nucleic acid apeared

first and they initiated the origin of genetic code (Kauffman, 1993 ).

Proteins First

The "protein first concept" drew its strength from the protenoids (proto-proteins

or protogenes). The plausibilty of this view comes from the view that small peptides

and polypeptides might have played a central role in early organisms( Longberg and

Gilbert, 1985). Calvin(l969) pointed out that a lage number of peptides in

contemporary bacteria and higher organisms are formed purely by enzymatic reactions,

where genetic code does not play any role. Further evolution ensured their information

translated into specific nucleotide sequence. The foremost problem with this concept

is that how the proteins were able to replicate itself to preserve information and evolve

further.

Nucleic Acid First

According to the "nucleic acid first concept" the biological information has evolved ab

initio from spontaneously formed polynucleotides ( protogenes ). The latter concept is

not supported by simulated experiments on prebiotic evolution (Dose, 1984 ). The co­

polymerization reaction with aspartic acid, glutamic acid and all other amino acids

respectively documented the wide scope of simple thermal copolymerization of a­

amino acids. The experiments done with aspartic acid, was part of a crucial conceptual

bridge to show thermal copolymerization of a-amino acids, including protenoids as one

20

of the many products. The emergence of ab initio concepts is also benefitted by thermal

copolymerization of amino acids ( Matsano, 1984 ). The spontaneatiy of the

appearance of nucleic acid, however, lack experimental support.

Synthesis of Energy Rich compounds

Protenoids and I or microspheres have been used as components of systems

that mediate other phenomena of envisioned prebiological significance. Lysine rich

protenoids promote the room temperature formation of aminoacyl adenylates from

amino acids and ATP, the later provides not only the adenylate moiety but also the·

energy. Aminoacyl adenylates serve as the starting materials and the energy source (as

observed in contemporary protein synthesis ) for polyamino amino acids, formed

especially in the presence of lysine-rich protenoid, oligoadenilic acid is also formed in

the experiment.

A mixture of lysine-rich protenoid and homo-polynucleotides results in

particulate associations that have been studied as models for primitive ribosomes.

Such complexes promote the formation of polyamino acids from the adenylates; the

kind of polyamino acid formed in greatest abundance can be under empirical

conditions, related in a codonic or an anticodonic way to the kind of

homo-polynucleotide complexed with protenoid(Rohling, 1984 ).

The origin of genetic information

The deoxyribonucleoside, from which DNA is assembled are more difficult to

21

deal with chemically than their ribose counterparts in RNA. The synthesis of

monomers of DNA in cells proceeds via ribose intermediates, and DNA replication

itself is initiated with RNA primers( Eigen et.al, 1982). In present day organisms

genetic information is processed by complex protein-RNA machinery. For such

machinery to have evolved, the information carriers themselves must have had

structural features that made them targets of recognition. Single-strand RNA can fold

to form a great variety of three dimensional structures, in contrast to uniform double

helix of DNA. In the present cellular machinery, where both functional and

instructional properties are required and they are found in RNA(Cech, 1989) . There

is no-reason to think it was otherwise during life's early stages. Nor is there any reason

to think there was a process whereby information stored in any other form could have

been transferred onto nucleic acids. The search for the likely chemical identity of the

genes points to RNA.

RNA strands with a homogeneous stereochemistry and with the correct

covalent bonding in the backbone of the strand could reproducibly lead to stable

secondary structures, or folding of the molecule, as a result of the formation of

hydrogen bonds between pairs of complementary nucleotides. This was an important

advantage , the 'Strands were more resistant to hydrolysis. The cleavage by a water

molecule is ultimate fate of polymers in solvated form. The primitive RNA

strands, that had the right backbone and the right nucleotides, had a crucial advantage.

They alone were capable of stable self-replication. They could

simultaneously act both as the source of instruction ( through the base pairing rules )

22

and the target molecules to be synthesise~ according to the instruction. This aspect is

highlited in the figure 5. Between function or the information neither could precede

the other, they had to evolve simultaneously.

The chemical species and processes of prebiotic times surely had a variety of

features in common with the present day biochemistry. As previously mentioned,

Sidney Fox and his colleagues( 19 56) have shown that the enzymatic functions can be

exercised by protenoid polymers made essentially by warming a mixture of amino acids.

In addition, to such primitive catalysts there were also other molecules which showed

stimulation by sunlight; they were lipids or lipid like molecules and they also could

form membranous structures. Even polysaccharides, or sugar polymers, were potential

source of energy. In short, a wealth of functional molecules would have been made

under pre-biotic environment through non-organic chemical paths (Eigen et.al, 1982)

The role of these functional molecules may have been vital in pre-biotic

chemistry. Their accidental efficiency to a specific reaction rested on non-accidental

structural constraints, such as favourable interactions with neighbouring molecules or

particular spatial folding. If there efficiency was to improve, and if more functional

· ones, they would have to escape such structural constraints. Only self replicative,

information conserving molecules could do so (Eigen et. al, 1982).

Self replication

The prebiotic molecular evolution is an essential stage in the sequence

of steps which lead to the emergence of self-replicating molecular mechanism,

23

Hexanucieotide template

d (3'o Phcl p GpGp Cp GpCpC Me5'). . . . . . . . . . . . .

., ._ . . "),.

r1 (5M<? rr Cp Gf!J) r1 < r:; c fl G r ·G r () P h ~ , 3 ,) d ( 5, M <l c p c p G p c p () p (, .:, ph c I )

Trideoxynucleotiae substrates

Figure 5 Aut<)catalytic r~plicatiQn of an RNA hqxamer which specif,cally aligns with and

ligat~s rts ~omponqnt trimqrs (From Joyce 1987)

preceding the evolution of the cell and life on earth. The synthesis and the availability

of basic subunits like amino acid and nucleic acid bases in prebiotic environment is

well accepted (Miller, 1953; Harda and fox, 1964). The established subsequent steps

for the formation of oligo-nucleotides and oligo-peptides in primordial soup are

supported from the simulated experiments (Lohramann and Orgel, 1973; Miller and

Orgel, 1974; Fox and Dose,1977; Lohramann et. al. 1980 ). It is important to

mention that in these studies ( Lohramann and Orgel, 1973; Lohramann et. al. 1980)

the activated ilnidazolide nucleotide bases do form RNA polymer, in the presence

of Zn ++ or Pb++ ions. The essence of these results, that it was possible to form poly­

nucleotide sequences from the activated nucleotide bases in the absence of enzyme

was an important step towards understanding the pre-biotic chemical evolution of

· molecules. At the same time, it is apparent that in the absence of any enzyme the

synthesis of poly-nucleotide sequences of RNA are limited to the size 20 to 40 base

long ( Lohramann and Orgel, 1973; Lohramann et. al. 1980 ). For self replication

machinery to evolve in primordial soup the information contents of RNA in the

absence of enzyme, in terms of the size of the genetic code, should be much larger

(Eigen et.al,1982).

Information Crisis

The availability of large information on RNA is crucial as only then the

primitive replk.ase enzyme could be translated. These inferences can be drawn from the

experiments done by Spiegelman ( 1967, 1971 ). In these experiments the virus Q~

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has 4,500 base long genome, part of which codes for the Q~-replicase enzyme, inside

the infected Escherichia coli. Important finding of this experiment was that the enzyme

Q~-rep1icase in the presence of the four energised substrate nucleotides: ATP, GTP,

UTP and CTP: even if no RNA template is present synthesises the RNA

( Spiegelman, 1967 and 1971 ). These results on one hand have shown that the

evolution of self-replication, in the presence of primitive replicase type of enzyme,

would have been the sequence preceding evolution of the cell (Eigen and Schuster,

197 8 ). But, on the other hand no explanation came forth, so far, to explain the

mechanism of RNA synthesis, in the absence of the enzyme, for the evolution of

information contents large enough to provide the code for a primitive replicase enzyme

system. As shown by above mentioned experiment the RNA of about 4,500 base long

is required to code for Q~-rep1icase enzyme (Spiegelman, 1971). This information

crisis is the crucial missing piece in the quest to explain prebiotic evolution of self

replicating molecular mechanism( Eigen and Schuster, 1978 ). This aspect, so far, has

been ignored as no convincing scientific theory has come forth to explain the evolution

of such a large information contents in RNA without the presence of an enzyme. It is

also important to note that in the absence of enzyme the laws of chemistry impose

constraints on the size of RNA, any further increase in the size will lead to the

hydrolysis. Only, if one assumes that this information crisis somehow was resolved,

only then, the rest of the steps leading to the emergence of the self replicating

machinery would fall in place. In this regard, the theory of hypercycle stands out as

an important contribution. A brief account of this theory is mentioned below.

26

Hyperrycle

The concept of hypercycle concerns with the prebiotic molecular evolution of

information carriers(RNA) through replication, in the presence of a premitive enzyme

system. As fidelity of information in the absence of an enzyme system is low ( 1 0-1 00

nucleotides base pair long), the system could evolve with the help of hypercyclic

linkages following, essentially, Darvvinian law of selection and survival, in the presence

of enzyme. The Orgel's experiments explained the increase in the size of RNA I 0-I 00

nucleotides long in the abence of the enzyme system. If the presence of the replicase

like enzyme is assumed then the RNA size could grow I 00-I 0000 nucleotides long

through hypercycle linkages.

Outline of The Proposed Theory in Present Thesis

In figure 6, the important stages related to the pre-biotic molecular evolution are

shown. The box representing the growth in information, of information bearing

molecules (RNA or DNA), is labeled with a question mark Till date no satisfactory

explanation has come forth to explain this lacunae in the theory of Origin of Life on

earth. In present work we propose to evolve a theory which beyond resonable doubt

explains the growth in information, in the absence of enzyme system. The theory is

based on thermodynamics of crowded solutions and takes into account fundamental

traits common to all life form. Chapter 2 provides the details of the assumptions as a

framework for the proposed theory which includes the thermodynamics of molecular

27

crowding and its consequences on the reaction kinetics. In chapter 3, the details of the

scaled particle theory (Reiss, 1 969) is provided. Methodology of calculations to

PRE-BIOTIC MOLECULAR EVOLUTION

Figure 6

PRE-BIOTIC CONDmONS

ENZYME FREE INF'OruvtATION GRO'W'I'H

?? . . PRESENT WORK

EMERGENCE OF SELF REPLICATION Theory of Hypercycle [ Eigen and Schuster]

*

evaluate the information growth of RNA type molecules under pre-biotic conditions

are given in chapter 4. This chapter also includes the calculations of dimensions of

small polypeptides and RNAmolecules by using Halle Molecular Graphics (HAMOG)

program. Results are given in chapter 5. The disscusion of the work is presented in

chapter 6 followed by the summary.

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