cell evolution · cell evolution remodeling by horizontal gene transfer 3 abstract the concepts of...

Cell Evolution Remodeling by Horizontal Gene Transfer

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Department of Biology Academic Year 2005-2006 Faculty of Sciences Lebanese University


Prepared by: Antoine Sawaya et al.

Supervised by: Prof. Dr. Fahd Nasr


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Table of Contents

Abstract 3

I. Introduction 3

II. Formation of the first cell 3

III. Cells in the RNA world 3

III.1. RNA-catalyzed polymerization 3

III.1.1. Mechanism of polymerization by protein polymerase 4

III.1.1.1. Substrate binding 4

III.1.1.2. Fidelity 4

III.1.1.3. Processivity 5

III.1.2. Technique of in vitro selection of ribozymes 5

III.1.2.1 General procedure 5

III.1.2.2. Selection of ligase ribozymes 5

III.1.3. Types of ribozymes catalyzing polymerization 6

III.1.3.1. Ligase ribozymes 6

III.1.3.2. Class I ligase 6

III.1.3.3. Hc ligase 7

III.1.3.4. Hairpin ribozyme 9

III.2. RNA-catalyzed translation 10

III.2.1. Mechanism 10

III.2.1.1. Steps 10

III.2.1.2. Factors affecting it 10

III.2.2. Action of ribozymes 11

III.2.2.1. Step 1 11

III.2.2.2. Step 2 12

III.2.2.3. Step 3 13

III.2.3. Uncatalyzed addition of amino acids on a template RNA 14

III.2.3.1. Technique 14

III.2.3.2. Results 14

III.2.4. Evolution of translation 15

III.3. RNA-membrane interactions 15

III.3.1. Introduction 15


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III.3.2. RNA9: RNA 9: RNA 10 complex 16

III.3.2.1. Structure 16

III.3.2.2. Function 16

III.4. RNA-protein interactions: riboswitches 17

III.4.1. Introduction 17

III.4.2. Types of riboswitches 17

III.5. Evolution in the RNA world 19

IV. Transition from RNA to DNA world 21

IV.1 Retropositioning 20

IV.2 Methyl-RNA 21

V. Discussion 22

Glossary 22

References 23


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Abstract The concepts of cell evolution are advancing in recent years with constant steps. It has im-

proved from being based on hypothetical theories to becoming increasingly founded on rigid

basis due to contemporary studies related to this issue. This report presents a coherent over-

view on the major transitions throughout evolution that have formed life as it is now. It propos-

es theories for the formation of the first cell, the dominance of RNA during a certain evolution-

ary period over other life forms, and the transition from RNA to DNA.

Keywords: RNA world, ribozymes, polymerization, translation, retropositioning.

I. Introduction Life has been defined as a chemical system capable of Darwinian evolution. So the first goal of

research into the origin of life has been to understand the first macromolecule capable of con-

taining information in the sequence of its monomer. With the emergence of the first atmos-

phere, environmental conditions have shaped the life on earth. First, the lack of ozone layer

allowed the entrance of UV light to the earth’s surface. This presented a selective pressure for

the formation of oligonucleotides mainly RNA. This led to a world dominated by RNA that

performed all the necessary functions required for survival such as: replication, translation, and

others. Not only did these RNA’s survive in such conditions but also they were able evolve

through horizontal gene transfer.

At a certain point in time, named by Carl Woese as “Darwinian Threshold”, a barrier

was established between the germ line and the somatic line. This, in addition to other processes,

was the primary factor that induced the formation of DNA from RNA and its dominance over

modern cells.

II. Formation of the First Cell Life began with the formation of a polymer having both catalytic capacity and the ability to

contain and propagate information. Charles Darwin proposed that the first protein evolved

from non-living matter. First the atmosphere was not as today. It was too hot for (O), (H) & (N)

to exist alone, so they combined to form CH4, NH3, H2O (vapor) & H2 …to make a reducing

atmosphere. There was also no free O to form the ozone layer to protect the earth from UV

light and electric storm and that was the incentive for the formation of amino acids (Oparin,


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1924). To prove this theory an experiment was done by Professor Urey and his student Stanley

Miller in 1950s.

C, N, O, and S (biogenic elements) in the interior stars have been ejected into the sur-

rounding interstellar medium. They were modified by a variety of physical and chemical pro-

cesses (UV, cosmic ray bombardment, gas phase chemistry and interstellar shock waves by

supernova). These modified C, N, O and S form at low temperature the dense molecular clouds

then form ice mantels (from H2O, CO, CO2, CH3OH, NH3, and others). The ice mantles are

exposed to ionizing radiation and form complex molecular species. A cycle of stellar birth and

death leads to the synthesis and evolution of organic matter that can be delivered intact to and

mixed with these products on planetary surfaces.

After amino acids have been created on earth, the earth began to cool (geographic rea-

sons), water vapor condensed and formed oceans, seas and lakes. As a consequence, simple

organic molecules began to accumulate and amino acids would have then polymerized in

clay/rock depressions.

Cell membrane is made up of amphiphilic products (membrane forming molecules con-

taining both polar and non polar groups on the same structure). Self assembly of organic mole-

cules in liquid water leads to the formation of the first membrane, and it occurs when small

amphiphlic molecules associate by hydrophobic interactions into more complex structures with

defined compositions and organizations. The hydrophobic phase of the cell membrane has the

potential to capture visible light energy that help in the evolution of cell membrane and protect

other components from damage by UV light. The polycyclic aromatic hydrocarbon in the me-

teorites (also defined as the primary constituent of the first cell membrane) helps in the stability

of the cell membrane (as cholesterol does in biological membrane). The study of first pre-biotic

self assembly was first investigated by microscopic gel structures called coacervates and

proteinoid microsphere.

III. Cells in the RNA World III.1. Ribozyme-Catalyzed Polymerization The process of ribozyme-catalyzed polymerization has always been a dilemma of the RNA

world theory. Scientists tried to define certain ribozymes that are able to undergo such process-

es, and they were able to distinguish some types.


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III.1.1. Mechanism of Polymerization by Protein Polymerases

Before defining these ribozymes, it is crucial to determine the mode of action of normal protein

polymerases during polymerization. This process is a simple attack of the 3’OH group of a

ribose on a phosphoanhydride bond of ATP to form 3’-5’ phosphodiester bond and a pyro-

phosphate molecule. This process involves 3 steps: substrate recognition and binding with the

help of metal ions, fidelity of polymerization, and processivity of the mechanism

III.1.1.1. Substrate binding

The ribozyme must bind to the template-primer complex and to the NTP’s to join them togeth-

er later on. This binding is mediated by charge-charge interactions, base (aromatic) stacking

interactions, and H-bonds (see figure 1). The active site of the ribozymes possesses conserved

negatively charged acidic residues that can bind divalent metal ions. These ions first function

in attaching the NTP’s and positioning them in the active site of the ribozyme to prepare them

for attack. As the NTP’s bind to the ribozyme, other positive charges bind to the phosphate-

ribose moieties on template-primer complex so that both substrates come in close proximity.

This allows H-bonds between NTP and templating base and base (aromatic) stacking interac-

tions between the primer and the NTP. The second function of the metal ions is the acid-base

catalysis. They stabilize the developing negative charges on transition states and provide strain

by distorting ground state more towards the transition state. This occurs by deprotonating the

3’OH of the ribose forming 3’O-. Furthermore, the formed O- is able to attack the α-phosphate

of the NTP forming 3’-5’ bond and releasing pyrophosphate.

In certain cases, ribozymes might utilize monovalent ions such as the hammerhead and

VS ribozymes. Moreover, nitrogenous bases in NTP’s might act in deprotonation of the 3’OH

instead of the divalent metal ions.

III.1.1.2. Fidelity

H-bonding between base pairs occurs at 3 sites between C and G and 2 sites between A and T;

these sites include O2 in pyrimidines and N3 in purines. Interactions of the polymerases with

these groups ensure high fidelity; these interactions are possible because upstream of the repli-

cation site, the B form RNA is changed to A form allowing opening of the RNA. Bending of

the primer-template complex allows polymerases to access a larger surface area for base pair

recognition.


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III.1.1.3. Processivity

It is the ability of a polymerase to form an oligonucleotide successively without dissociating

from the template. Ex: β-complex of DNA Polymerase II, PCNA complex in eukaryotes…

(McGinness and Joyce, 2003).

Figure 1. Mechanism of polymerization by protein polymerases. Addition of an NTP involves a two metal ion mechanism with no direct participation of the protein side chains. Electrostatic in-teractions involving divalent metal cations are indicated by dotted lines. Solid circles indicate metal-bound water molecules (McGinness and Joyce, 2003).

III.1.2. Technique of in vitro selection of Ribozymes, SELEX (Systematic Evolution of

Ligands by Exponential Enrichment)

III.1.2.1. General Procedure

It involves a population of DNA templates that could be of random sequence or complemen-

tary to a known ribozyme with various mutations. They are transcribed to form an RNA pool

or population by T7RNA polymerase. From this pool, a fraction of RNA molecules is selected

depending upon its ability to assume certain functions. These RNA’s are reverse transcribed by

reverse transcriptase to form cDNA that is amplified by PCR. This process is repeated for

many cycles for further selection of the needed RNA’s.

III.1.2.2. Selection of the Ligase Ribozymes

The SELEX mode is utilized for selecting ligase ribozymes. The RNA pool and primers are

added. The RNA’s consist of different 220 nucleotides with their 5’and 3’ ends being constant

regions. The 5’ constant end is formed of the template region and a hairpin used to anneal to

the primer placing it in a position where ligation or primer extension would occur (Komoto,

2000).


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III.1.3. Types of Ribozymes Catalyzing Polymerization

III.1.3.1. Ligase Ribozymes

They catalyze the ligation of the 3’ end of the primer to the 5’end of the ribozyme itself. They

are of many groups but the most important include class I ligase, L1 ligase, R3/R3C ligase, and

hc ligase.

III.1.3.1.a. L1 ligase

It forms 3’-5’ linkages, loses the 5’ hairpin that is added to all RNA’s, but base pairs with an

internal sequence from the 5’added template. It is allosterically controlled i.e. its function is

controlled by small molecule effectors that were evolved to protein effectors. Its motif is a 3-

way junction that catalyzes the ligation of the primer bound to one of the 3 stems. Since its

function can be regulated by proteins, it can be directly affected by environmental factors, a

fact that is essential in a fluctuating RNA world.

III.1.3.1.b. R3/R3C

R3: Its active site is of 74 nucleotides arranged in a 3-way junction motif with the primer bind-

ing at the 3’end. This variant lacks the C nucleotide and was able to catalyze ligation of the

primer to the 5’ end of the ribozyme; thus, even simple ribozymes (lacking C) could be able to

ligate primers. R3C: This variant possesses C, and its rate of catalysis is higher than the R3

type by 20-folds (McGinness and Joyce, 2003).

III.1.3.1.b. Dual Action Ribozymes

These are able to catalyze ligation as the other ligases and to catalyze self-cleavage at the same

time. This dual action occurs at different sites but is important since it shows the extent of evo-

lution and development of the RNA molecules especially needed in the RNA world (Burke,

2004).

III.1.3.2. Class I ligase

III.1.3.2.a. Ligation Type

The 5’ nucleotide of the ribozyme was positioned in a template-bound manner to mimic an

added NTP on a primer for primer extension. This is credited to the added template-hairpin

complex added at this end. As L1 ligase, this ribozyme loses it hairpin, utilizes the 5’ template


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region to bind the primer, and ligates the primer by 3’-5’ bond. What is important is that a sin-

gle template nucleotide was retained for base pairing with the 5’ nucleotide of the ribozyme.

III.1.3.2.b. Type able to undergo primer extension

(i) First type

The ligase ribozyme was divided between P1 and P3 stems, and the template and half of P2

were provided as a separate RNA molecule but can bind to the primer. The primer-template

complex was recognized by the ribozyme by base pairing allowing P2 stem to rejoin this com-

plex. As the restructured ribozyme is formed, it catalyzes the addition of 3NTP’s on the primer.

The fidelity of this process decreases to 85% as the amount of GTP increases since G can pair

with U in a mismatch named the GU wobble pair decreasing the efficiency of the reaction

(McGinness and Joyce, 2003).

(ii) Second Type

E278-19 Ribozyme: It was selected when the primers added to the medium lacked their last 2

nucleotides; as binding occurs, the ribozyme needed to catalyze the polymerization of these 2

nucleotides and then to undergo ligation. This ribozyme was able to add NTP’s in the 5’-3’ as

well as the 3’-5’ directions (McGinness and Joyce, 2003).

(iii) Third Type

The core catalytic domain of class I ligase was used. Many modifications were performed on it.

First, a random sequence of 76nt was added to 3’end, and 8 random nucleotides were inserted

at each of 2 locations within the ribozyme. Then, the 5’ end of the primer was ligated with the

5’ end of the ribozyme (to prevent sequence specific interactions). Next, the internal template

region was sequestered, and the template was added as a separate molecule. This formed a

class of ribozymes that no longer required base pair interactions of primer-template complex to

bind to the ribozyme, that worked in a sequence-independent manner, and that added 14 nucle-

otides onto the (10-60) nucleotide primer. Its fidelity is about 99% but decreased when GTP

was present at high concentration. Following a mismatch, this ribozyme stopped the polymeri-

zation process to prevent inacurate base pair additions. However, it is not self-replicative

(Burke, 2004).


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III.1.3.3. Hc ligase

III.1.3.3.a. Structure

It is formed from tetrahymena group I ribozyme; its main part is formed from the independent-

ly forming P4-P6 domain and the P3 and P8 stems slightly modified. Its primer-template com-

plex, the substrate, is helical and can base pair with the P1 stem of the ribozyme. This is helped

by interactions of the P4-P6 domain. This domain possesses 2A-A pairs in J4/5 region that are

able to interact with a GU wobble pair in P1. One A (A114) binds to G in 2’OH-2’OH bond;

the other, A207, interacts with N2 in G (2’OH-N2 bond). The U in GU controls the movement

of G; it orients G in a manner allowing it to form its bonds. To allow this ribozyme to undergo

intermolecular reactions, certain mutations must be inserted. This is risky since it might lead to

mutations inside the functional region; therefore, a new strategy was developed. A poly (U)

linker was added between the 5’end of the ribozyme and the 3’end of the stem-loop region, the

region that binds the helical substrate, allowing pseudo intermolecular ligation to occur. A

more evolved hc ligase is the 18-2 type that has 19 mutations with respect to the initial struc-

ture; some of these mutations occur in J4/5 and J7/8 regions of the P1-P4 complex (see figure

2).

III.1.3.3.b. Function on external template

Its main function is to ligate or add primers on an external template that could be an RNA hair-

pin or 2 linear primers complementary to the same substrate (template). It forms 3’-5’

phosphodiester bonds; while at high concentrations it binds with other ribozymes decreasing its

ligation efficiency. Ligation depends on the recognition of substrate-hairpin complex (helix),

its binding to P1 stem-loop region, and then the ligation of the 3’ end of the primer to the 5’end

of the ribozyme. The ligation efficiency increases with the presence of a GU wobble pair at

ligation site; beyond this site, any change of nucleotide sequence in the hairpin (template) of

the new complex reduces drastically ligation. This proves that the loop sequence of the hairpin

is crucial through its interaction with the ribozyme - for correctly orienting the substrate-

hairpin complex within the active site. 18-2 ribozyme binds stem portions of the hairpin-

substrate complex in a sequence-independent manner; this is extremely important to be able to

act on different RNA molecules. The last function detected for the 18-2 ribozyme is its ability

to add (2-3) nucleotide on the primer (McGinness and Joyce, 2002).


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Figure 2. (A) Wild-type hc ligase ribozyme that was used to generate the starting population of ribozymes, with insertion of a poly (U) linker between the hairpin substrate and the 5’ end of the ribozyme. (B) Evolved 18-2 ligase ribozyme, with mutations relative to the wild-type sequence shown in red (McGinness and Joyce, 2002).

III.1.3.4. Hairpin ribozymes

It is a small catalytic RNA derived from the minus strand of the satellite RNA of TRV. It

promotes cleavage forming 2 RNA fragments with 5’OH and 2’-3’ cyclic phosphate termini

or ligation of RNA fragments to increase length and complexity of RNA (see figure 3). At

low temperature, H-bonds are needed to stabilize RNA’s, less base pairing with the ribo-

zyme is required, sequence complementarity is reduced, and RNA cleavage is hindered.

Stabilization of HPR-substrate complex increases ligation; this occurs at low temperature

and requires freezing to induce the catalytically active conformation of HPR. However, the

temperature range for efficient ligation is narrow (-4,-12) ºC whereby below or above this

range, the process becomes less efficient. Furthermore, ligation is enhanced by freezing-

thawing cycles that would result in increasing the variability of RNA’s formed by dissocia-

tion and redistribution of RNA’s. HPR, similar to other ligases, forms 3’-5’ bonds (Vlassov

et al., 2004).


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Figure 3. (A) Structure of the hairpin ribozyme with separated enzyme and ligation substrates. Helical segments H1±H4 as well as ligation substrates 3-LS and 5-LS and the cleavage/ligation site are as indicated. Ligation substrates 3-LS and 5-LS were obtained by HPR-catalyzed cleav-age of an internally 32P-labeled oligonucleotide (LP), consisting of the normal HPR substrate se-quence extended with flanking sequences for use as primer bindings sites for the experiments (Vlassov et al., 2004).

III.2. RNA-Catalyzed translation:

III.2.1. Mechanism

III.2.1.1. Steps

It is of many steps that are mainly acyltransfers. It includes, as a first step, the attack of the

carboxylate group of the aa’s on the α-phosphate of an ATP to form aa-AMP complex. The

second step involves the attack of 2’or 3’ OH group of a tRNA base on the carboxylate group

to form aa-tRNA complex. The last step involves 2 aa-tRNA complexes to be in close proximi-

ty to induce peptide bond formation between the aa’s and the loss of an aa-tRNA bond (see

figure 4 below) (Burke, 2004).

III.2.1.2.Factors affecting it

III.2.1.2.a. Leaving Group Reactivity

Leaving groups are defined as the products released when 2 molecules join together ex: pyro-

phosphate produced when aa-AMP complex is formed. As the leaving group is stable, it is fa-

vorable for the reaction, and the acyl donors (amino acids) are said to be highly reactive. Sta-

bility is inversely proportional to reactivity i.e. if the leaving group is stable, it is unreactive

and vice versa. In general, leaving groups with low pKa (acidic) are more reactive than others.

There are many types of them, but the most important ones are four and include

phosphoanhydrides, thioesters, cyanomethyl esters, and ribose esters. Phosphoanhydrides are

the best due to their adenine residue that is kept hidden in the active site until the attack of the


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tRNA on aa-AMP to form aa-tRNA. The second type is the thioester ex: Acetyl CoA, which

has its conjugate base stabilized by its own polarity. In addition to that, sugar esters might act

as good leaving groups even though they are less reactive than others ex: aa-tRNA bond. Fi-

nally cynomethyl esters are used as leaving groups especially in ribozyme-catalyzed protein

synthesis.

III.2.1.2.b. Nucleophilicity of the attacking atoms

It is the ease with which nucleophiles (electron donors) attack other molecules. Nucleophilicity

increases with the increase of negative charge on the atom, with increase in electronegativity,

and with a simplification in structure to prevent steric hindering forces.

III.2.1.2.c. Electrophilicity of carbonyl C

The C=O is a polar group with partial positive charge on C and partial negative charge on O.

This enhances the carbon’s susceptibility to nucleophilic attack and is prominent in RNA's that

have H-bond donors and acceptors.

III.2.1.2.d. Charge Stabilization

It is crucial to prevent unfavorable burying of charged species and to position them in the ac-

tive site. These are negative charges that are mainly present on the carbonyl O in transition

state and then the leaving group after formation of products (Burke, 2004).

III.2.2. Action of Ribozymes

III.2.2.1. Step 1: aa + ATP aa – AMP + PPi

3- mercaptopropionic acid (3MPA) was used instead of a normal aa since 3 MPA-GMP-

ribozyme complex is more stable then aa-GMP-ribozyme complex. This is necessary in order

to be able to select the RNA’s able to undergo addition of 3MPA to GMP. Thus, during this

process, 3MPA binds to the 5’end of the ribozyme, which is mainly a GTP allowing the for-

mation of the needed complex. Then it is selected through its ability to form S-S bridges be-

tween an S- group in 3 MPA and an S- containing compound (Burke, 2004).

III.2.2.2. Step 2 aa-(any leaving group) + tRNA aa-tRNA + leaving group

If adenylates are used, the AMP part would have been the interacting portion of the aa-AMP

complex. It has been demonstrated that some ribozymes (R29) are able to bind to AMP at the


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OH group and to join the aa to them. This ribozyme is non specific, and thus is able to bind any

aa-AMP complex without specificity. With further selection processes, a more evolved RNA

was developed, RNA77. This is a highly specific ribozyme of 90 nucleotides that recognizes

mainly Phe and Tyr. In addition to adenylates, other leaving groups include ribose esters and

cyanomethyl esters. During the process of in vitro selection, some ribozymes were able to bind

aa-CME groups. One example is a ribozyme that transfers aa’s from small oligonucleotides

(tRNA-like) to their own 5’end by stabilizing the transition state through divalent metal ions.

They were also able to catalyze the opposite reaction, which involves the transfer of the aa

from aa-CME to the oligonucleotide. A second example involves ribozymes that undergo a 2-

step mechanism. Such ribozymes were extended by 70 nucleotides at their 3’end from the ini-

tial group of ribozymes. They were able to bind aa’s from aa-CME complex and to transfer

them to a tRNA. This process is induced by interactions between divalent metal ions and 29

nucleotide and stem-loop region in the ribozyme ex:ADO2 ribozyme (see figure 5). A third

group of ribozymes can directly bind to tRNA’s (this group is extended by 70 nucleotides on

5’end of the initial ribozyme). The ribozyme-tRNA hybrid is cleaved off at the 5’end to form

aa-tRNA with bond at 3’O of aa (Burke, 2004).

Figure 4. Stepwise acyl transfer in peptide synthesis. The initial activation step converts a phosphoanhydride (indicated by bracket 1) into a mixed carboxy-phosphoanhydride (bracket 3). Reaction with tRNA produces a ribose ester (bracket 4), then condensation with another amino acid produces the peptide product (bracket 5). A76 indicates the 3’ terminal nucleotide of aminoacyl-tRNA (Burke, 2004).


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Figure 5. Ribozyme-catalyzed aminoacylation of tRNA starting from CME-activated amino acid and proceeding through an acyl-ribozyme intermediate (Burke, 2004).

III.2.2.3. Step 3: Peptide Bond Formation

Through in vitro selection, 4 groups of ribozymes catalyzing this reaction were defined. The

first group includes ribozymes that can catalyze peptide bond formation only if they have a

5’NH2 terminal (aa bound to 5’ end) not 5’OH. They can use only ribose esters to catalyze this

reaction while the other groups use adenylate phosphoanhydrides only. The second group is

different from the others in that it uses biotin as aa and 5’-imidazole-substituted U’s in their

RNA sequences. Their mechanism is not yet elucidated but can be credited to the proximity

effect i.e. the degree of closeness of the 2 aa-tRNA complexes that favors this process. Another

type of ribozymes, a 29nt RNA29, forms di- and tripeptides from the Phe-AMP compounds.

Their mechanism involves addition of Phe at the 3’end; then 1 or 2 additional aa’s are bound.

These aa’s condense directly into peptides because of the proximity effect. Finally, the last type

of ribozymes uses a flexible linker with a disulfide bond and a Phe (at the 5’end); peptidyl

tRNA was given in form of N-biotinylated Met with aa-AMP’s. The selected ribozyme was

able to form dipeptides of different sequences (Burke, 2004).

III.2.3. Uncatalyzed Addition of aa’s on a Template RNA

III.2.3.1 Technique

This technique involves 3 constituents: the aa, given in the form of a minihelix ex: Ala-

minihelix), the RNA template strand, and the P-tRNA complex. The guide RNA was comple-

mentary to a part of the Ala-minihelix and to the tRNA.

III.2.3.2. Results

The combination of the 3 constituents leads to the formation of Phe-Ala dipeptide not Ala-Phe.

This was contributed to the high energy P- bonds that favor the reaction in the C-N direction

(opposite to modern protein synthesis). However, this dipeptide was not found when the RNA

template’s interaction with the minihelix and tRNA were disrupted. Thus, such template is


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found to be essential for protein synthesis in this experiment. Since the reaction proceeded in

the C-N direction, the dipeptide was bound to the tRNA allowing further addition of aa’s and

the formation of tripeptides. This experiment proves that protein synthesis could proceed with-

out the need of a ribozyme but essentially needs an RNA template (Tamura and Schimmel,

2004).

Figure 6. (A) Reaction of dipeptide synthesis using Phe-tRNAPhe, template oligonucleotides, and Ala-minhelix (B) TLC analysis of reaction products produced from mixing Phe-tRNAPhe and Ala-minihelix without (lane 1) and with (lane 2) the template oligonucleotide (5’U2A6UGGU3’); a mismatched template oligonucleotide (5’U2A6CCCC3’) was used for lane 3. (C) Trichloroacetic acid precipitation of nascently formed dipeptide. After a 2-h incubation, three different reaction mixtures were precipitated with trichloroacetic acid (Tamura and Scimmel, 2004).

III.4. Evolution of Translation

This issue has always been under extensive debate. Even though it is impossible for an RNA

world to have a complete ribosome, it has been suggested that certain ribozymes (as seen in the

previous section) were able to catalyze peptide bond formation. However, at the onset of this

synthetic pathway, the peptides formed had no importance in their own; they were mainly pro-

duced to enhance the structural stability of the ribozyme and to increase its functionality by just

binding to it. In addition to that, the peptides formed had no specific sequence. Therefore, the

only selective advantage for peptide synthesis was its ability to produce compounds that en-

hance stability of RNA and increase the amounts of function such RNA’s have.

Some of the most important consequences of the production of small proteins include

formation of hairpins (as in riboswitches) and tertiary and secondary structures required for


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specifc functions of RNA’s. As a result, early peptide synthesis led to the evolution of the RNA

world allowing RNA’s to become more susceptible to environmental conditions.

With protein-induced evolution, coded translation emerged. The onset of this develop-

ment is related to the peptidyl transferase activity that exhibits a selective advantage through

amplyfying the affinities of small proteins and increasing the specificity of peptide-RNA inter-

actions. This occurs by adding additional aa’s that might induce a folded structure more affine

for RNA (Noller, 2004).

III.3. RNA-Membrane Interactions

III.3.1. Introduction

For a cell to survive when put in any medium, it must be able to interact with the outer sur-

roundings and to respond to external stimuli. This is observed in modern cells in the form of

protein channels that allow exchange of molecules between intra- and extra- cellular media.

However, in the RNA world, such complex proteins were still absent, and thus, translation was

at its beginning. RNA molecules must have acted as channels during this era. Studies done on

RNA molecules have shown that some of these molecules have the ability to bind to phospho-

lipids bilayers (especially phosphatidylcholine ) under normal physiological conditions present

nowadays. Such RNA’s disrupt the membrane allowing the exchange of ions between media

and the release of vesicle-bound molecules from inside the cell to the surrounding medium.

III.3.2. RNA9: RNA9: RNA10 complex

III.3.2.1. Structures

Studies on RNA’s to determine their binding efficiency to membranes have determined the

presence of a complex RNA9:RNA9:RNA10 that efficiently binds to membranes. Its structure

(on figure 7) shows right hand loops of RNA’s 9 to help in RNA9: RNA9 dimerization, and the

left-hand loops for RNA9:RNA10 binding. Since these interactions occur in loops, they are

named as “kissing” loops.

III.3.2.2. Function

When RNA9 or RNA10 molecules were present alone, their binding to the membrane was not

detectable; however when mixed together, binding increases drastically proving that

supramolecular aggregates of RNA are formed and required for binding to a lipid membrane.

This also proves that the RNA9:RNA9:RNA10 trimer can bind to many other similar trimers


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forming large tail-like RNA chains joined to the membrane. Furthermore, studies have shown

that these RNA aggregates bind to edges of the membrane not to smooth surfaces. This is due

to many reasons. First, RNA aggregates prefer areas where the lipids and membranes are irreg-

ular allowing easier access of a bulky RNA to its ligand, dioleoylphosphatidylcholine. Second,

RNA’s at bent lipid regions might stabilize pores by stabilizing irregular lipid conformations;

this is possible since RNA’s stabilize disturbances in the hydrophobic internal regions of the

membrane during ion penetration thus increasing their permeability (Vlassov et al., 2001; Janas

and Yarus, 2003).

Figure 7. Structures of RNA9 and RNA 10. Constant sequences are shown in lowercase; ran-domized nucleotides are capitalized. Mutated positions and changes are shown on arcs outside the sequences. Regions important for complex formation are shown in larger bold type (Vlassov et al., 2001).

III.4. RNA- Protein Interactions, Riboswitches

III.4.1. Introduction

In modern cells, certain bacterial genes are clustered in operons that are controlled by one pro-

moter sequence. In some operons, in addition to promoter sequences, leader sequences are also

present. These sequences have been proven to be conserved throughout evolution and are

named as riboswitches since they change secondary structure depending on the protein bound

to them. Such sequence interconverts in general between 2 structures, the terminator and the

antiterminator, that induce respective processes. These simple interactions might have led to

the evolution of translation from uncoded synthesis to a precisely coded protein whose function

might be altered if only 1aa is misplaced.


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III.4.2. Types of riboswitches

RNA plays a significant role in regulating a diverse set of cellular processes in all life. One of

the recently discovered forms of genetic regulation by RNA is the riboswitch. This question

was the focus of numerous studies that resulted in the discovery of more than 20 classes of

riboswitches distributed across many species (table 1).

Table 1. Different types of riboswitches showing their structure (consensus sequence), function, and regulation.

III.5. Evolution in the RNA World

The primordial cells in this world were highly unstable and underwent continuous cycles of

cell division and horizontal gene transfer (HGT). HGT allowed nuons, any nucleic acid se-

quence, to be interchanged between different cells, thus, the genetic material at this time was

highly dynamic. This increased the pace of evolution since it improved the formation and se-

lection of advantageous traits, it enhanced RNA polymerization and translation by improving

RNA efficiency, and it was the primary source for increasing RNA-protein interactions.

With the dominance of HGT, daughter cells produced had unequal amounts of nuons.

To overcome this problem, 2 strategies were implemented. The first involved gene duplication;

the genome would be replicated in multiple copies preventing the loss of any nuon during cell

Name of operon Structure of Aptamer

Effector Protein

Function Control

ptsGHI See fig-ure 12 (A)

GlcT, en-zyme II

Encodes genes of PTS (phospho transfer system)

In presence of glucose : enzyme II adds P to glu-cose, GLcT binds to lead-er forms antiterminator, and translation occurs, vice versa (in absence)

S- box riboswitch See figure 12 (B)

SAM (S-adenosyl- methionine)

Regulation of S-metabolism

When SAM is present , it binds to anti-anti-terminator preventing translation and vice versa

Theophylline- re-sponsive riboswitch: constructed from the theophylline aptamer joined to communication module I-2

See fig-ure 12 (C)

Theophylline Controls translation initiation since it is present near RBS

When theophylline is pre-sent , slipping of the I-2 bridge occurs allowing clearing of RBS thus; initiation of translation RBS: Ribosomal binding site

Coenzyme B12 Riboswitches

See fig-ure 12 (D)

Vitamin B12 Controls formation of vitamin B12

When vitamin B12 is pre-sent, it binds to leader forming terminator and vice versa in absence


19

division. The second method uses the strategy of joining different nuons together in form of a

primitive “chromosome-like” structure, the first genome (nuonome). This nuonome had several

advantages. First, replication occurs once, and all nuons are replicated. Second, certain non-

replicating nuons can bind to the nuonome allowing them to be replicated. Thus, this was a

selective advantage that was further enhanced by forming a barrier between it, the nuonome

(germ line), and the individual nuons not bound to it (somatic line) (Brosius, 2002).

A B

C D Figure 12. (A) Proposed schematic model of the antiterminator and terminator structures of the ptsG leader mRNA (Schilling et al., 2004). (B) SAM-directed terminator-antiterminator riboswitch. Two alternative structures of S-box, the anti-antiterminator (Upper) and antiterminator (Lower) (Ephstein et al., 2003). (C) Secondary structure of translation initiation region of xylR gene with the riboswitch and H2-1 module. The part in the grey box is the module. The mechanism of slipping done by this aptamer is shown (Suess et al., 2004). (D) (A) Consen-sus sequence and secondary-structure for the metabolite-binding domain of B12 riboswitches; red nts are bases conserved by 90% (Nahvi et al., 2003).


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IV. Transition from RNA to DNA Even though such transition is crucial for evolution, the theories that try to explain it are still

under extensive study and debate. Many obstacles have been determined and not solved, ex:

the formation of ribonucleotide reductase and the emergence of reverse transcriptase (needed)..

Nevertheless, some theories have been postulated for this transition and two of these will be

discussed below.

IV.1. Retropositioning

This theory proposes that an early form of reverse transcriptase was able to form DNA from

RNA. The produced DNA segments were then integrated into the RNA genome by illegitimate

recombination forming a hybrid RNA-DNA genome. A very important feature of DNA is that

it is chemically more stable than RNA. The absence of the 2’OH group in DNA prevents the

self-cleavage process common in RNA whereby 2’OH attacks a nearby phosphate binding to it

and cleaving the phosphodiester bonds. Since genomes need to be stable for efficient transla-

tion and replication, the selective advantage of DNA was obvious. With evolution, DNA was

favored, and at a certain point in time, it became the genome.

The process of reverse transcription and then recombination is generally named as

retropositioning. Since the DNA retrogenes are formed from mRNA’s, they lack regulatory

elements: promoters and terminators. If, by chance, the newly produced genes are integrated

near promoter regions of other genes, these source genes would exhibit new functions that

could be advantageous for survival. In other cases, certain promoters are mobile elements and

can move from one gene to another. If they integrate inside the structural gene, they induce the

formation of new proteins that could be favorable for the cell.

Thus, in addition to being an important mechanism in the transition pathway from RNA

to DNA, retropositioning can generate new genes and functions providing variability in RNA

sequences, which is essential for evolution (Brosius, 2002).

IV2. Methyl-RNA

Another theory for this transition includes an intermediate genome, methyl-RNA. The assump-

tion involved in this theory is that the only pathway for forming DNA from RNA is through

ribonucleotide reductase enzymes. These enzymes are complex and undergo radical mecha-

nisms to change 2’OH to 2’H, which is impossible in the RNA world. Thus, it is clear that the

transformation of RNA to DNA cannot occur in one step.


21

It has been proven that certain enzymes, snoRNA’s and methylases, are able to methyl-

ate the 2’OH of ribose to form a methyl-RNA. This process has 2 advantages: the silencing of

2’OH that is generally self-cleaved and the prevention of intra-molecular H-bonding favoring

one tertiary structure over another. This stimulates variability in RNA structure and therefore

RNA function.

Furthermore, studies on snoRNA’s have shown that they existed in the RNA world and

that they might have catalyzed this reaction. However this occurred post-transcriptionally and

involved the methylation of specific sites leaving most of the RNA susceptible for cleavage.

Further studies have discovered certain ribozymes that can act as methylases and use S-

adenosyl-methionine (SAM) as a methyl donor. The mechanism involves positive charges in

SAM that attack H+ in 2’OH releasing it from O and forming O-. This O- acts as a nucleophile

and attacks CH3 to bind it forming 2’OCH3.

A third model for methylation describes a concerted action of snoRNA’s and

methylases. This method is important since it is nonspecific i.e. it could occur on any nucleo-

tide, and it allows post-replicative methylation as a means of stabilizing the newly produced

genome (Poole, et al. 2000).

V. Discussion It can be inferred that the study of cell evolution is extremely complex. It depends mainly on

recent studies that tend to simulate similar conditions that are thought to have existed in the

early history of earth. Therefore, the results obtained from such experiments are only predic-

tions of what would have happened at the onset of earth. However, this does not necessarily

mean that such predictions are not valid since they result from conditions that are known to

have been present in the ancient world. Further knowledge in this field requires efficient exper-

iments and techniques. This is concerned with molecular biology studies, and advances in this

field would surely unveil new secrets that might contradict all theories proposed nowadays.

Glossary Amphiphilic Products: membrane forming molecules containing both polar and non polar

groups on the same structure.

Electrophilicity: It is the tendency of certain molecules to gain electrons. Electrophiles are

positively charged.

Gene duplication: It is the formation of multiple copies of a certain gene.


22

Hairpin: It is a secondary form adopted by certain RNA’s that have palindromic sequences

(these are opposite in direction but complementary). These sequences bind to each other

forming hairpins.

Horizontal Gene Transfer: It is the transfer of genetic material between cells of the same

generation ex: sexual conjugation in prokaryotes.

Nucleophilcity: It is the tendency of certain molecules to attack positive nuclei. These are nec-

essarily electron donors that are negatively charged.

Phosphoanhydride Bond: It is the bond occurring between phosphate molecules in ATP.

PCR (Polymerase Chain Reaction): It is a technique that cleaves and separates DNA frag-

ments from each other.

Retroposition: It is the formation of RNA from DNA by reverse transcriptase and its integra-

tion in the genome.

Ribozyme: It is a catalytically active RNA molecule.

References 1. Brosius, J. (2003). Gene duplication and other evolutionary strategies: from the RNA world

to the future. Journal of Structural and Functional Genomics, 3: 1-17.

2. Burke, D. (2004). Ribozyme-catalyzed genetics. IN: The Genetic Code and the Origin of

Life. Edited by de Poulpana, L.R. Plenum Publishers.

3. Ephstein, V., Mironov, A.S., and Nuddler, E. (2003). The riboswitch-mediated control of

sulfur metabolism in bacteria. P.N.A.S. USA, 100: 5052-5056.

4. Janas, T. and Yarus, M. (2003). Visualization of membrane RNAs. RNA, 9: 1353-1361.

5. Komoto, J. (2000). Introduction to the RNA world. Edited by Gegenheimer, P. Online:

http://rnaworld.bio.edu/class/RNA/RNA00/RNA_World_toc.html

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istry and Biology, 10: 5-14.

7. McGinness, K.E. and Joyce, G.F. (2002). RNA-catalyzed RNA ligation on an external

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before the Beginning of Life: Selection of the First Oligonucleotide-like Polymer by UV

Light, BMC Evolutionary Biology, 3:12.

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spread genetic control elements in prokaryotes. Nucleic Acid Research, 32:143-150.


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10. Noller, H.F. (2004). The driving force for molecular evolution of translation. RNA, 10:

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tween RNA and DNA. Chemistry and Biology, 7: R207-R216.

12. Schilling, O., Langbein, I., Muller, M., Schmallisch, M.H., and Stulke, J, (2004). A protein-

dependent riboswitch controlling ptsGHI operon expression in Bacillus Subtilis: RNA

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2853-2864.

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14. Sun, L., Cui, Z., Gottlieb, R.L., and Zhang, B. (2002). A selected ribozyme catalyzing di-

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