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Chapter 21. The Genetic Code and Protein Synthesis

Chapter 21. The Genetic Code and Protein Synthesis

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Animal Physiology



that genes are DNA molecules themselves. Thus, it becomes necessary to discuss here the

organization of chromosomes in order to understand the chemical nature of genes.



21.1 THE ORGANIZATION OF THE CHROMOSOME

The chromosomes are regarded to control all kinds of biochemical and physiological activities of the

cell through tiny chemical units called genes. They replicate with precision during cell division and

gametogenesis and help in the transmission of characters from one generation to another. Although

chromosomes vary in number in different organisms, they maintain unique similarity in terms of their

physical and chemical organization.

The genetic material of all cells is contained in the chromosomes which have a complex

composition. The human chromosomes contain approximately 15 per cent DNA. 10 per cent RNA

and 75 per cent protein, but the genetic material is almost always double stranded DNA. The viruses

are the only exceptions which contain single stranded DNA and in certain cases the single stranded

RNA functions as the genetic material. The chromosomes of viruses and bacteria are circular whereas

in all other organisms the chromosomes are linear structures. A typical haploid chromosome of a

complex organism is a cylindrical structure composed of two identical units, the chromatids. The

chromatids are intimately twined around each other and each one is supposed to be containing about

8 fibrillar threads. Each fibril is composed of two double helices of DNA. Both the chromatids are

attached with each other through a common point, the centromere, which represents a constricted

region on the chromosome.

The chromosomes of complex organisms contain long stretches of non-informative DNA and

some segments of DNA exist in multiple copies along a single chromosome. Although each species

has a characteristic amount of DNA, the eukaryotes vary greatly in DNA content which is always

more than the prokaryotes. One picogram of DNA, when properly stretched, would be equivalent to

31 cm of DNA. The chromatids are comprised of chromatin material which is heavily stained with

uranyl acetate.

The chromatin is a viscous, gelatinous material which contains DNA, RNA, basic proteinshistones and non-histone proteins. Histone proteins and DNA are present in a fixed ratio of 1:1, but

the non-histone proteins always vary in different tissues. Histones are small basic proteins rich in

arginine and lysine, and bind intimately with DNA. There are four types of histones: H2A, H2B, H3,

H4, each of which are present in equimolar amounts. There are certain regions in the chromosomes

which remain condensed during interphase and early prophase. These are defined as heterochromatin

regions which are genetically inactive. Besides these regions, the remaining chromosome remains in a

non-condensed state and is called euchromatin.



What are Genes?

Cytogenetic observations suggested that the chromosomes contain DNA and that it is the hereditary

material. Mendel gave the idea of discrete factors as hereditary determinants which are inherited.

These ‘factors’ were later termed as genes and defined as particulate units arranged on a thread-like

chromosome.



The Genetic Code and Protein Synthesis



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Morgan’s work on Drosophila melanogaster, and later work with bacterial genetics on frequency

of recombination led to the formulation of a correct definition of gene. Thus the gene is defined as a

unit of recombination which cannot be subdivided by chromosomal breakage or crossing over and can

be separated from its neighbouring segments by a crossing over event. As our knowledge of the gene

advanced, it was discovered that the chromosomal unit functioning as the gene can undergo mutation

and thus affect the physiological function or expression. It was soon realized that the effectiveness of

a gene depends upon its relation to other neighbouring genes and as such there may be overlapping

regions of gene function. From the studies of mutation and mutagenic agents it has been suggested

that the unit of mutation could be much smaller than the functional unit, that is much smaller than the

unit of recombination.

From the mutation studies in Drosophila, it has been found that the so-called white-eye gene

exists in a number of alleles, hence a variety of mutations of this gene may give rise to various eye

colours. From this, it has been surmised that a gene can exist in many forms with a difference in

functions which account for distinct observable phenotypes.



21.2 REPLICATION OF DNA

From biochemical studies, it has been proved beyond doubt that DNA is the universal genetic

material of all forms of life except certain viruses. The building blocks of DNA have been described

in Chapter 1. In 1947, Chargaff demonstrated that DNA contains equal proportions of purine and

pyrimidine bases, so that adenine and thymine, and cytosine and guanine are present in equimolecular

proportions. These observations were quite significant which became clear from the elucidation of the

double helix model of DNA by Watson and Crick in 1953.

The double helix model consists of two twisted polynucleotide chains in which the deoxyribose

sugar units on adjacent nucleotides are linked with phosphodiester bonds to form a sugar-phosphate

backbone. The purine and pyrimidine bases project inwards perpendicular to the sugar-phosphate

backbone and are linked by hydrogen bonds. There is, however, a specific base pairing, occurring

between adenine and thymine and between cytosine and guanine (Fig. 21.1). The polynucleotide

chains are complementary to each other and the hydrogen bonds between the polynucleotide bases

stabilize the double helix, The hydrogen bonds, however, are sufficiently weak and capable of

breaking and reforming at room temperature. The genetic information in the DNA molecule depends

on the sequence in which the four bases are arranged along the polynucleotide chains. In the

complementary chains, the phosphate-sugar linkages run in the opposite directions. If in one chain,

the sugar-phosphate links go from a 3'-carbon to a 5'-carbon, then in the complementary chain the

sugar-phosphate linkages would run from a 5'- carbon to 3'-carbon.

The double helical structure of Watson and Crick suggests the manner in which the DNA

molecule can undergo replication. By a variety of ingenious experiments it has been shown that DNA

replication is semi-conservative, that is, each strand in the double helix serves as a template for the

synthesis of a new strand simultaneously, while the original strand remains intact in daughter cells for

several generations.



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Animal Physiology



Fig. 21.1



Watson and Crick model of the double helix of DNA. The two chains are held together by hydrogen bonding

between the bases.



Semi-conservative Replication of DNA

In 1958, Meselson and Stahl experimentally demonstrated in E. coli bacteria that DNA is replicated

through semi-conservative mechanism. The chromosome of E. coli is a continuous DNA molecule,

whereas in plants and animals the chromosomes are more complex in organization. Hence, the

process of replication can be precisely elucidated in E. coli. Meselson and Stahl grew the bacteria

initially in a medium containing labelled nitrogen (l5N). After growing them for several generations, it

was found that all the DNA in bacteria was labelled. This was called heavy DNA. Then the bacteria

with heavy DNA were grown in an ordinary medium for one generation only. Analysis showed that

the next generation consisted of an intermediate form comprising of one heavy and one normal strand

of DNA. When the bacteria were grown in the ordinary medium, in the next generation, half of the

DNA was normal and another half of the intermediate form synthesized on the heavy strand

(Fig. 21.2).



The Genetic Code and Protein Synthesis

15 N



15 N



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15 N



14 N



15 N 14 N



G1



15 N



14 N



14 N 14 N



14 N 14 N



15 N 14 N



G2



Fig. 21.2



The mechanism of semi-conservative replication of DNA.



The in vitro synthesis of DNA was demonstrated by Kornberg in 1956 and he showed that DNA

polymerase catalyzes the replication of DNA molecule by addition of deoxyribonucleotides to the

free —OH group at the 3' end of a chain. The enzyme has two outstanding properties. Firstly, it

requires a mixture of the four types of DNA nucleotides (ATP, CTP, GTP and TTP) to function, and

secondly, a DNA primer which acts as template. In the absence of DNA primer no DNA synthesis

could occur.



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Animal Physiology



dTPPP

DNA

dGPPP

+ DNA primer

(–dTP –dGP –dAP –dCP)n

dAPPP

Polymerase

+ 4 (n) PP

dCPPP

It was found that DNA polymerase acts on the strand which is copied in the direction from 3' to

5' end. Since there are two strands in each DNA molecule, it is envisaged that the two strands unwind

which run in opposite directions so that the replication begins on each of the unwound strand.

Experimental evidence suggests that both strands are copied at the same time. In that case, the strand

which runs from 3' end to 5' end can be copied continuously. The other strand which runs in the

opposite direction from 5' to 3' end is copied in the opposite direction, in the 3' to 5' direction. The

synthesis cannot be continuous, hence newly synthesized DNA is formed in short segments which are

later joined by DNA ligase as discovered by Khorana and his associates.

We have seen that the parent DNA molecule unwinds before replication takes place but the

molecule does not unwind completely. Crick has pointed out that replication and unwinding take

place simultaneously. As soon as unwinding takes place, the formation of two new chains starts. The

mechanism is outlined in Figure 21.3.





5¢ 3¢







5¢ 3¢



5¢ 3¢



¢

5 3¢





























































C



Fig. 21.3



3 ¢ 5¢

B









A



Mechanism of discontinuous replication of DNA. (a) partial unwinding of the two chains showing synthesis at

the 3¢ end by the action of DNA polymerase. (b) newly synthesized DNA is in short length chains. (c) the new

chains are joined by DNA ligase and reform new helixes.



The Genetic Code and Protein Synthesis



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The rate of DNA replication in E. coli is a fast process, but in higher organisms it is rather slow.

In mammals, since the chromosome replication includes histone synthesis as well, the process is much

slower. At the same time, autoradiographic studies show that in eukaryotic chromosome, there are

several initiation points.



21.3 THE GENETIC CODE

While proposing the double helix model of DNA. Watson and Crick postulated that DNA is the

information system in which the bases were incorporated in a manner so as to determine the sequence

of amino acids in a protein molecule. This hypothesis visualizes that DNA utilizes a language of four

letters, adenine, guanine, cytosine and thymille, in different combinations. Through these four bases

DNA utilizes information to be transmitted to the cytoplasm for cellular control and its functions. But

then, this code has three variants in the form of DNA, tRNA and mRNA. In the information theory,

it was suggested that the code is operationally a system of codes and anticodes and if one was

deciphered the other one could be automatically determined.

In the DNA molecule, there is a mutual attraction for adenine and thymine, and for cytosine and

guanine which make the base pairs. DNA acts as the template for RNA synthesis. In the transcription

of RNA the attractive forces would be between adenine and uracil since RNA does not contain

thymine. Only one strand of double-helical DNA can act as a template for RNA synthesis. All types

of RNA molecules (rRNA, tRNA and mRNA) are single stranded and complementary to the DNA

template.

The messenger RNA (mRNA) contains a linear sequence of bases which dictates the sequence of

amino acids of all polypeptide chains. The sequence of bases on mRNA is known as the genetic code.

There are twenty different kinds of amino acids and therefore there must be a specific code for each

amino acid. It was proposed by George Gamov that a combination of three bases seems to be most

probable which would yield 64 different combinations (43). In 1964, Khorana synthesized a

messenger RNA of known sequences though which he determined the triplet-base sequences that

could code different amino acids. The codes on the mRNA are called codons specific for each amino

acid.

There are a few generalizations about the genetic code (see Table 21.1). Several of the amino

acids have more than one codon, hence redundancy is present in the codes. For example, leucine is

coded by at least six codons, CUA, CUU, CUC, CUG, UUA, and UUA. The code is commaless and

universal for all protein synthesizing organisms. There are three codons, UAA, UGA and UAG which

do not code for any amino acid hence, specify termination of a peptide chain. The code is nonoverlapping.



21.4 SYNTHESIS OF POLYRIBONUCLEOTIDES

RNA molecules differ from DNA in two respects. RNA is a long-chain molecule in which there is

ribose sugar instead of deoxyribose and the fourth base thymine is replaced by uracil. Experimental

evidence indicates that RNA is synthesized iron a DNA template by a process analogous to the

replication of DNA and this process is known as transcription.



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Animal Physiology



Table 21.1

First

base



U



C



A



G



The Genetic Dictionary: Scheme for the Genetic Code

Second

base U



G



Third

base



C



A



phenylalanine

phenylalanine

leucine

leucine



serine

serine

serine

serine



tyrosine

tyrosine

terminate

terminate



cysteine

cysteine

terminate

tryptophane



U

C

A

G



leucine

leucine

leucine

leucine



proline

proline

proline

proline



histidine

histidine

glutamine

glutamine



arginine

arginine

arginine

arginine



U

C

A

G



isoleucine

isoleucine

isoleucine

methionine



threonine

threonine

threonine

threonine



asparagine

asparagine

lysine

lysinee



serine

serine

arginine

arginine



U

C

A

G



valine

valine

valine

valine



alanine

alanine

alanine

alanine



aspartic acid

aspartic acid

glutamic acid

glutamic acid



glycine

glycine

glycine

glycine



U

C

A

G



A specific enzyme called DNA-directed RNA polymerase is required to catalyze the synthesis of

RNA on DNA templates. Like DNA polymerase, this enzyme also requires nucleoside triphosphates

which align themselves on one of the DNA strands. When RNA synthesis starts, the DNA strands

unwind. In vitro synthesis of RNA has revealed that the enzyme RNA polymerase is Mg2+ dependent

and requires appropriate nucleoside triphosphates and a DNA template. Synthesis of RNA starts from

the 5' end of the RNA chain, hence in this respect it resembles DNA polymerase. Chemical analysis

has shown that RNA is copied as single strand from only one strand of the DNA molecule. Hence, the

newly synthesized RNA strand should be complementary to the DNA strand which is copied. This

has been amply proved with the help of hybridization experiments.

While a strand of RNA is synthesized on DNA template, the RNA molecule rapidly detaches

itself and the two unwound DNA strands come together after the reformation of hydrogen bonds. The

rate of formation of the phosphodiester bonds is rather slow (9000 per minute at 37°C) which is

suggestive of a number of starting points along the DNA chain. It has been experimentally proved

that a number of RNA molecules can be transcribed simultaneously on a DNA template. This shows

that the enzyme RNA polymerase can attach to DNA at a number of places. The growth of RNA

chains takes place from 5' to 3' direction as in case of DNA.

It is now known that RNA polymerase has a complex structure and functions on signals when to

start synthesis of RNA and when to stop. A factor sigma (s) gives the signals to start RNA synthesis

and it is an integral part of the enzyme molecules as such. The termination message to stop synthesis

of RNA is given by a protein factor rho (r) which is not considered as part of the polymerase.

As a general rule, RNA is formed on a DNA template. However, there are some exceptions to

this general rule, for example, viruses whose genetic information is contained in a single RNA strand,



The Genetic Code and Protein Synthesis



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must first replicate a complementary RNA strand which may be copied to produce more. This

synthesis is carried out by RNA-directed RNA polymerase.

According to the central dogma in molecular genetics the genetic information is transferred from

DNA to RNA only. But in 1970, Temin reported that in some cancer producing RNA viruses copies

of DNA are produced on RNA templates by an RNA-dependent DNA polymerase, called reverse

transcriptase. The newly synthesized DNA may be incorporated in the host cell chromosome or it

may be used to synthesize more of viral RNA. This discovery generated tremendous interest among

the workers and the possible role of viruses in inducing cancer in humans was postulated.

Nevertheless, it is not necessary that the presence of the reverse transcriptase activity may be the

cause of malignancy of the virus to the host cells. Normal cells have also been shown to contain

reverse transcriptase activity, which according to some, may play some role in gene amplification

during differentiation of amphibian oocytes, and accidently the enzyme may produce new DNA

sequences which may lead to malignant cancerous growth.



Function of Ribonucleic Acids

RNA is always found as a single stranded structure but the molecule may occur as secondary and

three dimensional structures which are no doubt related to their functions. RNA is found to occur in

several species as described below.



Transfer RNA

Transfer RNA (tRNA) is also referred as soluble RNA or adaptor RNA. The molecules are relatively

small, about 75-85 nucleotides long. In a prokaryotic cell (E. coli), there are at least 60 different

species of tRNA, while in eukaryotic cells, there may be as many as 100 tRNA molecules. Hence

there may be two or three or more species of tRNA which can bind a single amino acid. Its molecular

weight is about 25,000 to 30,000 The RNA chain is bent upon itself and this brings the compatible

bases close to each other through hydrogen bonding. As a result, the tRNA molecule assumes a

cloverleaf pattern (Fig. 21.4) which relates to its secondary structure. Another special feature of tRNA

molecule is that it possesses a number of unusual bases.

A sequence of 3 bases is found on the middle lobe of the tRNA which is complementary to the

triplet code on messenger RNA. All tRNA have a terminal base sequence cytidylic - cytidylic adenylic acids (CCA) at the 3' terminal. The terminal nucleotide adenylic acid residue serves as an

attachment site to the energy-rich enzyme-bound amino acid. Such an amino acid is attached to the

2nd or 3rd carbon of the ribose sugar of the terminal nucleotide. At the 5' end of the strand, the tRNA

has an unpaired base guanine. The middle lobe with the sequence of three bases is known as the

anticodon and the base sequence of this varies with the type of tRNA. The amino acyl-tRNA complex

enters the ribosome and with the help of anticodon triplet recognizes its corresponding codon on the

mRNA molecule. Thus the function of tRNA is to carry various amino acids from the pool and

arrange them in the ribosomes as required by the messenger RNA.



Ribosomes and Ribosomal RNA

The ribosomes are submicroscopic particles occurring in every living cell (70 S ribosomes in

prokaryotes and 80 S ribosomes in eukaryotes). These are the sites for protein synthesis which are



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Animal Physiology



Fig. 21.4



The cloverleaf pattern of a typical tRNA molecule.



present throughout the cytoplasm in bacteria, whereas in higher organisms they are found attached to

the endoplasmic reticulum. About 60 per cent of the ribosome is RNA and the remaining 40 per cent

is protein. The ribosome is formed by the union of two subunits—a smaller 30 S* subunit, and a

larger 50 S subunit. The smaller subunit has one large ribosomal RNA (rRNA) which is 16 S

consisting of 1,600 nucleotides and 21 different proteins. The larger subunit has one large RNA (23

S) consisting of 3,200 nucleotides and 34 different proteins, and another smaller RNA (5 S) having

120 nucleotides. The task of ribosomal RNA is to join up the sequence of amino acids brought on the

assembly line of the messenger RNA molecule to form a peptide chain.

Both the 30 S and 50 S subunits are heterogenous mixture of three groups of proteins viz., unit,

marginal and fractional proteins. The ribosomes, owing to the existence of heterogeneous proteins in

them, serve different functions and fall under different classes. Some of these types of proteins are

known for their participation at various steps during protein synthesis. The 50 S subunit, in addition

to the above three groups, has another type of protein known as functional repeat (FR) protein.

rRNA is mainly synthesized in the nucleolus on extrachromosomal DNA as template. rRNA acts

as the framework on which the ribosomal particles are assembled during biosynthesis of ribosomes.

They maintain ribosomal particles in a configuration permitting them to fulfill their role in protein

biosynthesis.

*S is a Svedberg unit at which a particle sediments in a high speed ultracentrifuge.



The Genetic Code and Protein Synthesis



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Messenger RNA

In 1961, Jacob and Monod demonstrated that the species of RNA which carries genetic information

from DNA to the ribosomes is the messenger RNA (mRNA). It has a thread like configuration which

may be coded for protein synthesis in small segments. It is synthesized enzymatically on DNA

template whose base sequence is complementary to DNA. In bacteria, messenger RNA has a short

life span, probably about 2 minutes, which is evident from the rapid turn over of viral proteins when

a phage infects the bacterium. The eukaryotic mRNA appears to be more stable, its half-life ranging

from a few hours to perhaps weeks.

Isolation of mRNA is a difficult process because of its short life and small quantity available in

the cell. However, techniques have been devised to isolate globin mRNA which synthesizes largely

haemoglobin from rabbit reticulocytes. The globin mRNA sediments at a small peak 9 S whose

estimated length is about 700 nucleotides. Messenger RNA has a base sequence complementary to

DNA of the same cell. This has been demonstrated by DNA-RNA hybridization experiments. Out of

the total-700 nucleotides of the globin mRNA, only 589 are coded by the DNA and the rest are

attached to the mRNA after transcription to its tail end. The globin molecule (protein) consists of 146

aminoacids, hence it would require only 436 nucleotides on the mRNA chain. Thus it is evident that

the mRNA has some extra nucleotides.

The prokaryotic mRNA is synthesized on one of the two strands of DNA acting as the template

and functions as a template for protein synthesis. However, the eukaryotic mRNA has two noncoding

regions which do not synthesize proteins. These regions are located at the 5' end and the 3' end. The

5' end has about 50 nucleotides whereas the 3' end has about 100 nucleotides. In some mRNAs the 3'

end is extraordinarily large.



21.5 PROTEIN SYNTHESIS

We have briefly described the structure and role of different types of nucleic acids. DNA is the

informational molecule which contains the genetic information in a coded form and this information

is transcribed on the mRNA molecules before it is translated by ribosomes for the synthesis of

specific proteins. Protein synthesis is thus a gene directed process which is carried out in the

cytoplasm utilizing an elaborate protein-synthesizing machinery. For convenience, the polypeptide

(protein) synthesis is discussed here under four stages: activation of amino acids; chain initiation;

chain elongation; and chain termination. As far as possible, a brief comparison will also be made

between protein synthesizing processes occurring in prokaryotes and eukaryotes to illustrate

fundamental differences.



Amino Acid Activation

There are 20 different types of amino acids in the cytoplasm of the cell forming an amino acid pool

and these are picked up one by one for assembly in a polypeptide chain. Activation of amino acid is

a prerequisite for its attachment to the specific tRNA molecule. Each one of them is selected and

bound by a specific enzyme, amino-acyl tRNA synthetase each of which is specific for each amino

acid. Each of the enzyme-bound amino acid reacts with ATP from the cytoplasm and forms amino



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Animal Physiology



Fig. 21.5



Steps in the activation of an amino acid.



acid adenylate-enzyme complex (Fig. 21.5). The enzyme-bound activated amino acid would then

readily react to bind with its specific tRNA. There is an amino acid specific for each tRNA, therefore

there will be 20 different amino acid tRNA complexes.

The activating enzyme has two separate catalytic sites. The activation reaction occurs in two

steps. The first reaction involves a reaction between ATP and the amino acid resulting in the

formation of aminoacyl adenylic acid and pyrophosphate along with AMP. The carboxyl terminal of

the amino acid binds with the 5'-phosphate group of the AMP through anhydride linkage (Fig. 21.5).

The second step involves the transfer of aminoacyl group to the tRNA molecule releasing adenylic

acid free.



Initiation of the Chain

The process of translation has to ensure that translation does not commence in the middle of mRNA

chain and also correct alignment of the first anticodon with the first triplet codon of the messenger

RNA. In case of wrong alignment the reading frame will be shifted to cause frame-shift mutations.

Hence, chain initiation follows a series of events and includes a process of complex formation in the

ribosome.

The following components are involved:

1. mRNA chain.

2. A special tRNA known as methionyl tRNA (fmet-tRNA).

3. Three protein factors that initiate protein synthesis: IF1, IF2 and IF3.

4. Guanosine trophosphate (GTP).

The process of protein synthesis is perhaps best understood in E. coli, therefore the description

presented here would pertain to this organism unless otherwise stated. It has been well established in



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