BCHM 309: NUCLEIC ACID AND NUCLEOTIDE METABOLISM

Introduction

Nucleotides are the Building Blocks of the Nucleic Acids - DNA and RNA.

Nucleotides perform a wide variety of functions

Building blocks for nucleic acids
 Universal energy carriers (ATP, GTP)
 Activators (e.g. UDP-glucose)
 Components of signal transduction pathways (cAMP, cGMP)
Nucleotides contain
 Ribose or deoxyribose sugar
 One to three phosphate groups
 purine or pyrimidine hetercyclic nitrogen base.

Cells Make Nucleotides by Two Pathways - de novo and Salvage Synthesis.

Purines are Made Separately from Pyrimidines

De novo verses salvage

SALVAGE PATHWAY

Activated ribose (PRPP) + base

Nucleotide

DE NOVO PATHWAY

Activated ribose (PRPP) + Amino acids + ATP + CO2

Nucleotide

1
Activation of Ribose-5-Phosphate

The ribose-5-phosphate portion of the nucleotide is generated from glucose in the pentose phosphate
pathway. To be used in nucleotide biosynthesis, ribose-5-phosphate needs to be activated by transfer of
pyrophosphate (PPi) from ATP to carbon 1 of the pentose. The activated sugar used is 5-
phosphoribosyl-1-pyrophosphate, PRPP. PRPP is generated by the action of PRPP synthetase.

Since the purines/pyrimidines are synthesized as the ribonucleotides, (not as the free bases) a
necessary prerequisite is the synthesis of the activated form of ribose 5-phosphate. Ribose 5-
phosphate reacts with ATP to form 5-Phosphoribosyl-1-pyrophosphate (PRPP). Conversion of
ribose 5-phosphate to 5-phosphoribosyl-1-pyrophosphate (PRPP)—a necessary step for the de
novo and salvage pathways of purine and pyrimidine biosynthesis

This reaction occurs in many tissues because PRPP has a number of roles - purine and pyrimidine
nucleotide synthesis, salvage pathways, NAD and NADP formation.

- A salvage pathway is a pathway in which nucleotides (purine and pyrimidine) are

synthesized from intermediates in the degradative pathway for nucleotides. Nucleotide
salvage pathways recover bases and nucleosides, from RNA and DNA degradation or from
exogenous sources, to convert them back to nucleotides. Nucleic acids are hydrolyzed to
their nucleotides by a variety of nucleases. Various nucleotidases and phosphatases further
breakdown into nucleosides. A third hydrolysis step by nucleosidases and nucleoside
phosphorylases release the constituent bases.

Salvage pathways are used to recover bases and nucleosides that are formed during degradation of
RNA and DNA. This is important in some organs because some tissues cannot undergo de novo
synthesis. Salvage reactions convert free purine and pyrimidine bases into nucleotides.

2
The salvaged bases and nucleosides can then be converted back into nucleotides.

- Purines and pyrimidines can be synthesized from smaller precursors (de novo synthesis).

Salvaging Purines

The more important of the pathways for salvaging purines uses enzymes called
phosphoribosyltransferases (PRT):

PRTs catalyze the addition of ribose 5-phosphate to the base from PRPP to yield a nucleotide.:

Base + PRPP = Base-ribose-phosphate (BMP) + PPi

3
Salvaging Pyrimidines

In pyrimidine salvage reactions, nucleosides and free bases generated by DNA and RNA
breakdown are converted back to nucleotide monophosphates, allowing them to re-enter the
pathways of pyrimidine biosynthesis and interconversion. PRPP serve as a source of ribose 5
phosphate.

Summary of Nucleotide biosynthesis

De novo synthesis

de novo pathway (anew; from scratch): nucleotides are constructed from simple precursors. Using
5-phosphoribosyl-1-pyrophosphate (PRPP), the de novo pathway enzymes build purine and
pyrimidine nucleotides from “scratch” using simple molecules such as CO2, amino acids and
tetrahydrofolate. This route of nucleotide synthesis has a high requirement for energy as compared
that of the salvage pathway

4
De novo synthesis of purine

Steps of de novo synthesis of purine

1. Purine Biosynthesis starts with the formation of an activated ribose intermediate. Ribose is
activated by the enzyme Ribose-5-phosphate Pyrophosphokinase. An older name for the enzyme
is 5-Phosphoribosyl-1-pyrophosphate Synthase. This enzyme catalyzes the transfer of
pyrophosphate from ATP to the anomeric hydroxyl group, the hydroxyl group on carbon one
ribose-5-phosphate. The product of the reaction is 5-phosphoribosyl-α1-pyrophosphate (PRPP)
and AMP.
The de novo synthesis of purines occurs in an interesting manner: The atoms forming the purine
ring are successively added to ribose-5-phosphate; thus, purines are directly synthesized as
nucleotide derivatives by assembling the atoms that comprise the purine ring system directly on
the ribose
2. Glutamine-PRPP Amidotransferase transferring the amide nitrogen of glutamine to the
anomeric carbon, C1 of 5-phosphoribosyl-1-pyrophosphate (PRPP).

3. Glycine (C4, C5, and N7) is added to N9 of the growing purine by glycinamide ribonucleotide
synthetase (GAR synthetase)
4. A formyl group (C8) donated by N10-formyl-tetrahydrofolate is attached to the free amino
group
5. Glutamine now donates its amido group (N3) to the carbonyl carbon.
6. The five-membered ring of the purine nucleus is closed by the enzyme AIR Synthetase.
7. CO2 (C6) is added to the growing purine ring system.

5
8. The amino group of aspartate is now linked to the just added carboxyl group.
9. The intermediated is cleaved releasing fumarate. The amino group of aspartate is left behind
and it becomes N1 of the purine.
10. A formyl group (C2) from N10-formyl-tetrahydrofolate is now added.
11. In the last step the 6-membered ring of the purine nucleus is closed by the action of IMP
Cyclohydrolase. The product is INOSINE-5´-MONOPHOSPHATE (IMP).

6
7
IMP is the precursor to both AMP and GMP. These major purine nucleotides are formed via
distinct two-step metabolic pathways that diverge from IMP

Control of Purine Biosynthesis

This pathway is controlled at several points.
 IMP, AMP, and GMP are allosteric effectors for Ribose-5-phosphate Pyrophosphokinase.
When their concentrations are elevated, the activity of this enzyme is inhibited.
 The Committed Step for purine biosynthesis is the reaction catalyzed by Glutamine-PRPP
Amidotransferase. This enzyme is allosterically inhibited by IMP, AMP, and GMP.

 The pathways from IMP to AMP and from IMP to GMP are also allosterically controlled.
AMP inhibits theactivity of Adenylosuccinate Synthetase. XMP and GMP inhibit the
action of IMP Dehydrogenase

8
De novo pathway of pyrimidine

1. The pathway for the formation of pyrimidine nucleotides begins with the formation of
carbamoylphosphate. This reaction is catalyzed by Carbamoylphosphate Synthetase II. This
enzyme takes glutamine as the ammonia donor, HCO3–, and 2 ATP molecules and catalyzes the
formation of carbamoylphosphate. Other products include glutamate, 2 ADP and a phosphate. This
enzyme is different from Carbamoylphosphate Synthetase I used for urea synthesis.
Carbamoylphosphate Synthetase II is a cytosolic enzyme requiring glutamine as the nitrogen
donor, whereas Carbamoylphosphate Synthetase I is a mitochondrial enzyme that
utilizes ammonia.

2. Carbamoylphosphate is then condensed with a molecule of aspartate to form

carbamoylaspartate. This reaction is catalyzed by the enzyme Aspartate Transcarbamoylase.

3. Dihydroorotase catalyzes a dehydration reaction that results in closure of the pyrimidine ring.
Dihydroorotate is the product of this reaction.

4. The dihydroorotate is oxidized to orotate by Dihydroorotate Dehydrogenase. In bacteria, the

enzyme is a NAD-linked flavoprotein containing bound FAD, FMN and Fe-S centers. In
eukaryotes, the enzyme is bound to the inner mitochondrial membrane. The electrons are
immediately accepted by a quinone (a CoQ like molecule) and then passed to the ET/OxPhos
pathway for ATP generation.

5. Orotate is now coupled to PRPP to form Orotidine-5´-monophosphate (OMP). This reaction is

catalyzed by Orotate phosphoribosyl Transferase.
6. In the last step of the pathway OMP is decarboxylated by OMP Decarboxylase to form Uridine-
5´- monophosphate (UMP).

9
  Synthesis of UTP from UMP
UMP is converted to UTP in two step Kinase reaction with 2 molecules of ATP
 UMP + ATP UDP + ADP
 UDP + ATP UTP + ADP
 Synthesis of CTP from UMP

The UTP is then converted to Cytidine-5´-triphosphate (CTP) by CTP Synthetase. This enzyme
takes the amide group from glutamine and attaches it to the carbonyl carbon, C4, of UTP. The
hydrolysis of ATP drives the reaction to completion. The other product is glutamate

10
 Control of Pyrimidine Biosynthesis
Pyrimidine nucleotide biosynthesis is controlled at the step catalyzed by Carbamoyl phosphate
Synthetase II. This is an allosteric enzyme. PRPP and ATP activate the enzyme and UDP and
UTP are allosteric inhibitors of its activity.

Synthesis of deoxyribonucleotides from ribonucleotide

This can be achieved by reduction of their corresponding ribonucleotide at position C2 of ribose
sugar catalyse by ribonucleotide reductases.

Nuclease
any enzyme that cleaves nucleic acids. Nucleases, which belong to the class of enzymes called
hydrolases, are usually specific in action, ribonucleases acting only upon ribonucleic acids (RNA)
and deoxyribonucleases acting only upon deoxyribonucleic acids (DNA). Some enzymes having
a general action (such as phosphoesterases, which hydrolyze phosphoric acid esters) can be called
nucleases because nucleic acids are susceptible to their action. Nucleases are found in both animals
and plants.
Nucleotides with Specific Functions
Nucleotides with specific functions include ATP. NAD, FAD+ and Coenzyme A
1. Adenosine triphosphate or ATP is often called the energy currency of the cell because this
molecule plays a key role in metabolism, particularly in energy transfer within cells. The molecule
acts to couple the energy of exergonic and endergonic processes, making energetically unfavorable
chemical reactions able to proceed.

11
The key to energy production lies with the phosphate groups. Breaking the phosphate bond is an
exothermic reaction. So, when ATP loses one or two phosphate groups, energy is released. More
energy is released breaking the first phosphate bond than the second.

ATP + H2O → ADP + Pi + Energy (Δ G = -30.5 kJ.mol-1)

ATP + H2O → AMP + PPi + Energy (Δ G = -45.6 kJ.mol-1)

The energy that is released is coupled to an endothermic (thermodynamically unfavorable)

reaction in order to give it the activation energy needed to proceed.

2. Nicotinamide adenine dinucleotides, NAD and NADP, are indispensable cofactors involved in
several redox reactions in all forms of cellular life.

3. In biochemistry, flavin adenine dinucleotide (FAD) is a redox-active coenzyme associated

with various proteins, which is involved with several important enzymatic reactions in
metabolism. A flavoprotein is a protein that contains a flavin group, this may be in the form of
FAD or flavin mononucleotide (FMN).

3. Coenzyme A (CoASH) has a clearly defined role as a cofactor for a number of oxidative and
biosynthetic reactions in intermediary metabolism.

Therefore, Nucleotides are important constituents not only of RNA and DNA, but also of a number of
key biomolecules considered many times in our study of biochemistry. NAD+ and NADP+, coenzymes
that function in oxidation-reduction reactions, are metabolites of ATP.

Diseases associated with Abnormal Nucleotide Metabolism

Lesch-Nyhan syndrome is a rare inborn error of purine metabolism characterized by the absence
or deficiency of the activity of the enzyme hypoxanthine-guanine phosphoribosyltransferase
(HPRT). Purines are nitrogen-containing compounds found in many foods (e.g., organ meats,
poultry, and legumes). In the absence of HPRT, the purines hypoxanthine and guanine are not built
into nucleotides. Uric acid levels are abnormally high in people with Lesch-Nyhan syndrome and
sodium urate crystals may abnormally accumulate in the joints and kidneys. Lesch-Nyhan
syndrome is inherited as an X-linked recessive genetic disorder that, with rare female exceptions,

12
most often affects males. The symptoms of Lesch-Nyhan syndrome include impaired kidney
function, acute gouty arthritis, and self-mutilating behaviors such as lip and finger biting and/or
head banging. Additional symptoms include involuntary muscle movements, and neurological
impairment.

DNA, the Genetic Material

DNA, deoxyribonucleic acid, is the genetic material in your cells. It was passed on to you from
your parents and determines your characteristics. The discovery that DNA is the genetic material
was another important milestone in molecular biology.

Griffith Searches for the Genetic Material

Many scientists contributed to the identification of DNA as the genetic material. In the 1920s,
Frederick Griffith made an important discovery. He was studying two different strains of a
bacterium, called R (rough) strain and S (smooth) strain.

 He injected the two strains into mice. The S strain killed (virulent) the mice,
 but the R strain did not (non-virulent) (see Figure below).
 Griffith also injected mice with S-strain bacteria that had been killed by heat. As expected,
the killed bacteria did not harm the mice.
 However, when the dead S-strain bacteria were mixed with live R-strain bacteria and
injected, the mice died.

13
Griffith’s Experimental Results. Griffith showed that a substance could be transferred to harmless
bacteria and make them deadly.

Based on his observations, Griffith deduced that something in the killed S strain was transferred
to the previously harmless R strain, making the R strain deadly. He called this process
transformation, as something was "transforming" the bacteria from one strain into another strain.
What was that something? What type of substance could change the characteristics of the organism
that received it?

DNA Replication

Replication is the process by which a double-stranded DNA molecule is copied to produce two
identical DNA molecules. DNA replication is one of the most basic processes that occurs within
a cell. Each time a divide, the two resulting daughter cells must contain exactly the same genetic
information, or DNA, as the parent cell. To accomplish this, each strand of existing DNA acts as
a template for replication.

Key points:

 DNA replication is semiconservative. Each strand in the double helix acts as a template
for synthesis of a new, complementary strand.
 New DNA is made by enzymes called DNA polymerases, which require a template and
a primer (starter) and synthesize DNA in the 5' to 3' direction.
 During DNA replication, one new strand (the leading strand) is made as a continuous
piece. The other (the lagging strand) is made in small pieces.
 DNA replication requires other enzymes in addition to DNA polymerase, including DNA
primase, DNA helicase, DNA ligase, and topoisomerase.

Starting DNA replication

How do DNA polymerases and other replication factors know where to begin?

 Replication always starts at specific locations on the DNA, which are called origins of
replication and are recognized by their sequence.
 Helicase is the first replication enzyme to load on at the origin of replication. Helicase's
job is to move the replication forks forward by "unwinding" the DNA (breaking the
hydrogen bonds between the nitrogenous base pairs).
 Proteins called single-strand binding proteins coat the separated strands of DNA near the
replication fork, keeping them from coming back together into a double helix.
 One new strand, which runs 5' to 3' towards the replication fork, is the easy one. This strand
is made continuously, because the DNA polymerase is moving in the same direction as the
replication fork. This continuously synthesized strand is called the leading strand.
 The other new strand, which runs 5' to 3' away from the fork, is trickier. This strand is made
in fragments because, as the fork moves forward, the DNA polymerase (which is moving

14
away from the fork) must come off and reattach on the newly exposed DNA. This tricky
strand, which is made in fragments, is called the lagging strand.

The small fragments are called Okazaki fragments, named for the Japanese scientist who
discovered them. The leading strand can be extended from one primer alone, whereas the
lagging strand needs a new primer for each of the short Okazaki fragments.

Summary of Replication.

15
Note:
DNA gyrase: structure and function. ... DNA gyrase is an essential bacterial enzyme that
catalyzes the ATP-dependent negative super-coiling of double-stranded closed-circular DNA

GENE EXPRESSION
Introduction
Central Dogma
DNA codes for protein not directly but indirectly through the processes of transcription and
translation. The indirect route of information transfer is known as the central dogma of molecular
genetics

DNA RNA Protein

The term dogma means “set of beliefs”; it dates from the time the idea was put forward first as a
theory. Since then the “dogma” has been confirmed experimentally, but the term persists.
The central dogma is the vital principle of molecular genetics because it summarizes how the
genetic information in DNA becomes expressed in the amino acid sequence in a polypeptide chain.
The sequence of nucleotides in a gene specifies the sequence of nucleotides in a molecule of
messenger RNA; in turn, the sequence of nucleotides in the messenger RNA specifies the sequence
of amino acids in the polypeptide chain.

Gene Expression
 gene is a small section of DNA that contains the instructions for a specific molecule,
usually a protein.
 The purpose of genes is to store information.
 Each gene contains the information required to build specific proteins needed in an
organism.

Gene expression is the process through which information from a gene is used in the synthesis of
a functional gene product. These products are often proteins, but in non-protein coding genes such
as transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.

The process of gene expression involves two main stages:

Transcription: the production of messenger RNA (mRNA) by the enzyme RNA polymerase,
and the processing of the resulting mRNA molecule.

Translation: the use of mRNA to direct protein synthesis, and the subsequent post-translational
processing of the protein molecule.

16
Some genes are responsible for the production of other forms of RNA that play a role in translation,
including transfer RNA (tRNA) and ribosomal RNA (rRNA).

The main concept in the central dogma is that DNA does not code for protein directly but
rather acts through an intermediary molecule of ribonucleic acid (RNA). The structure of RNA
is similar to, but not identical with, that of DNA. There is a difference in the sugar (RNA contains
the sugar ribose instead of deoxyribose), RNA is usually single-stranded (not a duplex), and RNA
contains the base uracil (U) instead of thymine (T), which is present in DNA. Actually, three types
of RNA take part in the synthesis of proteins:

A molecule of messenger RNA (mRNA), which carries the genetic information from DNA and
is used as a template for polypeptide synthesis. In most mRNA molecules, there is a high
proportion of nucleotides that actually code for amino acids.

Several types of ribosomal RNA (rRNA), which are major constituents of the cellular particles
called ribosomes on which polypeptide synthesis takes place.

A set of transfer RNA (tRNA) molecules, each of which carries a particular amino acid as well
as a three-base recognition region that base-pairs with a group of three adjacent bases in the
mRNA. As each tRNA participates in translation, its amino acid becomes the terminal subunit
added to the length of the growing polypeptide chain. The tRNA that carries methionine is denoted
tRNAMet, that which carries serine is denoted tRNASer, and so forth.

Gene control regions

 Start site. A start site for transcription.

 A promoter. A region of few hundred nucleotides 'upstream' of the gene (toward the 5'
end). It is not transcribed into mRNA, but plays a role in controlling the transcription of
the gene. Transcription factors bind to specific nucleotide sequences in the promoter region
and assist in the binding of RNA polymerases.

In eukaryotes like humans, the main RNA polymerase in the cells does not attach directly
to promoters like bacterial RNA polymerase. Instead, helper proteins called basal
(general) transcription factors bind to the promoter first, helping the RNA polymerase
in the cells get a position on the DNA.

Many eukaryotic promoters have a sequence called a TATA box. It's recognized by one of
the general transcription factors, allowing other transcription factors and eventually RNA
polymerase to bind.

 Enhancers. Some transcription factors (called activators) bind to regions called 'enhancers'
that increase the rate of transcription. These sites may be thousands of nucleotides from
the coding sequences or within an intron. Some enhancers are conditional and only work
in the presence of other factors as well as transcription factors.

17
  Silencers. Some transcription factors (called repressors) bind to regions called 'silencers'
that depress the rate of transcription.


TRANSCRIPTION
The process of making an RNA strand from a DNA template is transcription, and the RNA
molecule that is made is the transcript. The manner in which genetic information is transferred

from DNA to RNA. The DNA opens up, and one of the strands is used as a template for the
synthesis of a complementary strand of RNA. The base sequence in the RNA is complementary
(in the Watson–Crick pairing sense) to that in the DNA template, except that U (which pairs with
A) is present in the RNA in place of T

Transcription initiation
To begin transcribing a gene, RNA polymerase binds to the DNA of the gene at a region called
the promoter. Basically, the promoter tells the polymerase where to "sit down" on the DNA and
begin transcribing.

Each gene (or, in bacteria, each group of genes transcribed together) has its own promoter. A
promoter contains DNA sequences that let RNA polymerase or its helper proteins attach to the
DNA. Once the transcription bubble has formed, the polymerase can start transcribing

Elongation
Once RNA polymerase is in position at the promoter, the next step of transcription—elongation—
can begin. Basically, elongation is the stage when the RNA strand gets longer, thanks to the
addition of new nucleotides.
During elongation, RNA polymerase "walks" along one strand of DNA, known as the template
strand, in the 3' to 5' direction. For each nucleotide in the template, RNA polymerase adds a
matching (complementary) RNA nucleotide to the 3' end of the RNA strand.

18
The RNA strand looks similar to DNA, except that it contains the base uracil in place of thymine
and has ribose sugars (which have a hydroxyl group on the 2' carbon) in place of deoxyribose
sugars.

RNA polymerase synthesizes an RNA transcript complementary to the DNA template strand in
the 5' to 3' direction. It moves forward along the template strand in the 3' to 5' direction, opening
the DNA double helix as it goes. The synthesized RNA only remains bound to the template strand
for a short while, then exits the polymerase as a dangling string, allowing the DNA to close back
up and form a double helix.
The RNA transcript is nearly identical to the non-template, or coding, strand of DNA. However,
RNA strands have the base uracil (U) in place of thymine (T), as well as a slightly different sugar
in the nucleotide. So, as we can see in the diagram above, each T of the coding strand is replaced
with a U in the RNA transcript.
Transcription termination
RNA polymerase will keep transcribing until it gets signals to stop. The process of ending
transcription is called termination, and it happens once the polymerase transcribes a sequence of
DNA known as a terminator.

Termination in bacteria

There are two major termination strategies found in bacteria: Rho-dependent and Rho-
independent.
In Rho-dependent termination, the RNA contains a binding site for a protein called Rho factor.
Rho factor binds to this sequence and starts "climbing" up the transcript towards RNA polymerase.

19
Rho-dependent termination. The terminator is a region of DNA that includes the sequence that
codes for the Rho binding site in the mRNA, as well as the actual transcription stop point (which
is a sequence that causes the RNA polymerase to pause so that Rho can catch up to it). Rho binds
to the Rho binding site in the mRNA and climbs up the RNA transcript, in the 5' to 3' direction,
towards the transcription bubble where the polymerase is. When it catches up to the polymerase,
it will cause the transcript to be released, ending transcription.
When it catches up with the polymerase at the transcription bubble, Rho pulls the RNA transcript
and the template DNA strand apart, releasing the RNA molecule and ending transcription. Another
sequence found later in the DNA, called the transcription stop point, causes RNA polymerase to
pause and thus helps Rho catch up.4^44start superscript, 4, end superscript
Rho-independent termination depends on specific sequences in the DNA template strand. As
the RNA polymerase approaches the end of the gene being transcribed, it hits a region rich in C
and G nucleotides. The RNA transcribed from this region folds back on itself, and the
complementary C and G nucleotides bind together. The result is a stable hairpin that causes the
polymerase to stall.

Rho-independent termination. The terminator DNA sequence encodes a region of RNA that folds
back on itself to form a hairpin. The hairpin is followed by a series of U nucleotides in the RNA

20
(not pictured). The hairpin causes the polymerase to stall, and the weak base pairing between the
A nucleotides of the DNA template and the U nucleotides of the RNA transcript allows the
transcript to separate from the template, ending transcription.
In a terminator, the hairpin is followed by a stretch of U nucleotides in the RNA, which match up
with A nucleotides in the template DNA. The complementary U-A region of the RNA transcript
forms only a weak interaction with the template DNA. This, coupled with the stalled polymerase,
produces enough instability for the enzyme to fall off and liberate the new RNA transcript.
TRANSLATION
During translation, which is the second major step in gene expression, the mRNA is "read"
according to the genetic code, which relates the DNA sequence to the amino acid sequence in
proteins Each group of three bases in mRNA constitutes a codon, and each codon specifies a
particular amino acid (hence, it is a triplet code). The mRNA sequence is thus used as a template
to assemble—in order—the chain of amino acids that form a protein.

Within all cells, the translation machinery resides within a specialized organelle called the
ribosome. In eukaryotes, mature mRNA molecules must leave the nucleus and travel to the
cytoplasm, where the ribosomes are located. On the other hand, in prokaryotic organisms,
ribosomes can attach to mRNA while it is still being transcribed. In this situation, translation
begins at the 5' end of the mRNA while the 3' end is still attached to DNA.

In all types of cells, the ribosome is composed of two subunits: the large (50S) subunit and the
small (30S) subunit (S, for svedberg unit, is a measure of sedimentation velocity and, therefore,
mass). Each subunit exists separately in the cytoplasm, but the two join together on the mRNA
molecule. The ribosomal subunits contain proteins and specialized RNA molecules—specifically,
ribosomal RNA (rRNA) and transfer RNA (tRNA).

What is a genetic code?

The genetic code links groups of nucleotides in an mRNA to amino acids in a protein. In
translation, the sequence of nucleotides in the mRNA is "translated" into a sequence of amino
acids in a polypeptide. Genetic code is therefore defined as the set of rules by which information
encoded in genetic material (DNA or RNA sequences) is translated into proteins (amino acid
sequences) by living cells. The genetic code is a set of three-letter combinations of nucleotides
called codons, each of which corresponds with a specific amino acid or stop signal. Specifically,
the code defines a mapping between tri-nucleotide sequences called codons and amino acids; every
triplet of nucleotides in a nucleic acid sequence specifies a single amino acid.

21
Genetic code and codon

CHARACTERISTICS OF THE GENETIC CODE

1. Triplet nature:

 Singlet and doublet codes are not adequate to code for 20 amino acids; therefore, it was
pointed out that triplet code is the minimum required.

2. Degeneracy

 The code is degenerate which means that the same amino acid is coded by more than one
base triplet.
 Degeneracy does not imply lack of specificity in protein synthesis.
 It merely means that a particular amino acid can be directed to its place in the peptide
chain by more than one base triplets.
 For example, the three amino acids arginine, alanine and leucine each have six
synonymous codons.
 The code degeneracy is basically of 2 types: partial and complete.
 In partial degeneracy, the first two nucleotides are identical but the third (i.e., 3′ base)
nucleotide of the degenerate codon differs; for example, CUU and CUC code for leucine.
 Complete degeneracy occurs when any of the 4 bases can take third position and still
code for the same amino acid; for example, UCU, UCC, UCA and UCG all code for
serine.

3. Non-overlapping

22
  The genetic code is nonoverlapping, i.e.,the adjacent codons do not overlap.
 A nonoverlapping code means that the same letter is not used for two different codons. In
other words, no single base can take part in the formation of more than one codon.

4. Commaless

 The genetic code is commaless (or comma-free). There is no signal to indicate the end of
one codon and the beginning of the next.
 There are no intermediary nucleotides (or commas) between the codons.

5. Universality

 Universality of the code means that the same sequences of 3 bases encode the same
amino acids in all life forms from simple microorganisms to complex, multicelled
organisms such as human beings.

6. Polarity

 The genetic code has polarity, that is, the code is always read in a fixed direction, i.e., in
the 5′ → 3′ direction.
 It is apparent that if the code is read in opposite direction (i.e., 3′ → 5′), it would specify
2 different proteins, since the codon would have reversed base sequence.

STEPS INVOLVED IN TRANSLATION

Translation occurs in a structure called the ribosome, which is a factory for the synthesis of
proteins. The ribosome has a small and a large subunit and is a complex molecule composed of
several ribosomal RNA molecules and a number of proteins. Translation of an mRNA molecule
by the ribosome occurs in three stages: initiation, elongation, and termination.

During initiation, the small ribosomal subunit binds to the start of the mRNA sequence. Then a
transfer RNA (tRNA) molecule carrying the amino acid methionine binds to what is called the
start codon of the mRNA sequence. The start codon in all mRNA molecules has the sequence
AUG and codes for methionine. Next, the large ribosomal subunit binds to form the complete
initiation complex.

During the elongation stage, the ribosome continues to translate each codon in turn. Each
corresponding amino acid is added to the growing chain and linked via a bond called a peptide
bond. Elongation continues until all of the codons are read.

Lastly, termination occurs when the ribosome reaches a stop codon (UAA, UAG, and UGA).
Since there are no tRNA molecules that can recognize these codons, the ribosome recognizes that
translation is complete. The new protein is then released, and the translation complex comes apart

23
KEY POINTS

 Prokaryotic gene expression is primarily controlled at the level of transcription.

 Eukaryotic gene expression is controlled at the levels of epigenetics, transcription, post-
transcription, translation, and post-translation.
 Prokaryotic gene expression (both transcription and translation) occurs within the
cytoplasm of a cell due to the lack of a defined nucleus; thus, the DNA is freely located
within the cytoplasm.
 Eukaryotic gene expression occurs in both the nucleus (transcription) and cytoplasm
(translation).

Prokaryotic versus Eukaryotic Gene Expression

To understand how gene expression is regulated, we must first understand how a gene codes for a
functional protein in a cell. The process occurs in both prokaryotic and eukaryotic cells, just in
slightly different manners.

Prokaryotic organisms are single-celled organisms that lack a defined nucleus; therefore, their
DNA floats freely within the cell cytoplasm. To synthesize a protein, the processes of transcription
(DNA to RNA) and translation (RNA to protein) occur almost simultaneously. When the resulting
protein is no longer needed, transcription stops. Thus, the regulation of transcription is the primary

24
method to control what type of protein and how much of each protein is expressed in a prokaryotic
cell. All of the subsequent steps occur automatically. When more protein is required, more
transcription occurs. Therefore, in prokaryotic cells, the control of gene expression is mostly at the
transcriptional level.

Eukaryotic cells, in contrast, have intracellular organelles that add to their complexity. In
eukaryotic cells, the DNA is contained inside the cell’s nucleus where it is transcribed into RNA.
The newly-synthesized RNA is then transported out of the nucleus into the cytoplasm where
ribosomes translate the RNA into protein. The processes of transcription and translation are
physically separated by the nuclear membrane; transcription occurs only within the nucleus, and
translation occurs only outside the nucleus within the cytoplasm. The regulation of gene expression
can occur at all stages of the process. Regulation may occur when the DNA is uncoiled and
loosened from nucleosomes to bind transcription factors (epigenetics), when the RNA is
transcribed (transcriptional level), when the RNA is processed and exported to the cytoplasm after
it is transcribed (post-transcriptional level), when the RNA is translated into protein (translational
level), or after the protein has been made (post-translational level).

Prokaryotic vs Eukaryotic Gene Expression: Prokaryotic transcription and translation occur

simultaneously in the cytoplasm, and regulation occurs at the transcriptional level. Eukaryotic gene
expression is regulated during transcription and RNA processing, which take place in the nucleus,
and during protein translation, which takes place in the cytoplasm. Further regulation may occur
through post-translational modifications of proteins.

Post Transcriptional Modifications

The primary transcript formed undergoes modification in eukaryotes, namely splicing, tailing and
capping. In this way, post-transcriptional processing helps increase the efficiency of protein
synthesis by allowing only specific protein- coding RNA to go on to be translated. A modification
also takes place at the opposite end of the RNA transcript.

25
1.CAPPING- the 5′ end of primary transcript is attached with methylguonosine with the help of
enzyme guanosyl transferase. The cap protects the 5' end of the primary RNA transcript from
attack by ribonuclease.

2.SPLICING-the introns (regions of RNA that do not code for proteins) are cut apart from exons
(regions of RNA that do code for proteins) and the exons are again joined together. these process
us catalysed by spliceosome assembled from proteins and small nuclear RNA molecules that
recognize splice sites in the primary transcript.

3.TAILING-the 3′ end of primary transcript is added a adenylate and not a single adenylate but
many adenylate also known as polyA tail. It is catalysed by an enzyme poly adenylate
polymerase (PAP)

AFTER these 3 steps, primary transcript is converted into m-RNA. and transferred to ribosome
for translation

RNAs from eukaryotes undergo post-transcriptional modifications including: capping,

polyadenylation, and splicing. These events do not occur in prokaryotes. mRNAs in prokaryotes
tend to contain many different genes on a single mRNA meaning they are
polycystronic. Eukaryotes contain mRNAs that are monocystronic. Termination in prokaryotes

26
is done by either rho-dependent or rho-independent mechanisms. In eukaryotes transcription is
terminated by two elements: a poly(A) signal and a downstream terminator sequence.

Viral Replication

A virus is a small parasite that cannot reproduce by itself. Once it infects a susceptible cell,
however, a virus can direct the cell machinery to produce more viruses. Most viruses have either
RNA or DNA as their genetic material. The nucleic acid may be single- or double-stranded. The
entire infectious virus particle, called a virion, consists of the nucleic acid and an outer shell of
protein.

As viruses are obligate intracellular pathogens they cannot replicate without the machinery and
metabolism of a host cell. Although the replicative life cycle of viruses differs greatly between
species and category of virus, there are six basic stages that are essential for viral replication.

1. Attachment: Viral proteins on the capsid or phospholipid envelope interact with specific
receptors on the host cellular surface. This specificity determines the host range (tropism) of a
virus.

2. Penetration: The process of attachment to a specific receptor can induce conformational

changes in viral capsid proteins, or the lipid envelope, that results in the fusion of viral and cellular
membranes. Some DNA viruses can also enter the host cell through receptor-mediated
endocytosis.

3 . Uncoating: The viral capsid is removed and degraded by viral enzymes or host enzymes releasing the
viral genomic nucleic acid.

4. Replication: After the viral genome has been uncoated, transcription or translation of the viral
genome is initiated. It is this stage of viral replication that differs greatly between DNA and RNA
viruses and viruses with opposite nucleic acid polarity. This process culminates in the de novo
synthesis of viral proteins and genome.

5. Assembly: After de novo synthesis of viral genome and proteins, which can be post-
transrciptionally modified, viral proteins are packaged with newly replicated viral genome into
new virions that are ready for release from the host cell. This process can also be referred to as
maturation.

6. Virion release: There are two methods of viral release: lysis or budding. Lysis results in the
death of an infected host cell, these types of viruses are referred to as cytolytic.

27
Fig: stages for viral replication

Reverse transcriptase

reverse transcription is the process in cells by which an enzyme makes a copy of double stranded
DNA (complementary DNA , cDNA ) molecules are made from a single stranded RNA. The
enzyme that makes the DNA copy is called reverse transcriptase and is found in retroviruses,
such as the human immunodeficiency virus (HIV). Normal transcription involves the synthesis of
RNA from DNA; hence, reverse transcription is the reverse of this

In viruses, reverse transcriptase allows the virus to insert its DNA to the host cell's DNA, forcing
the cell to make more viruses. This is good for the virus but bad for the host.

Reverse Transcriptase in Viruses

In viruses, reverse transcriptase allows the virus to insert its DNA to the host cell's DNA, forcing
the cell to make more viruses. This is good for the virus but bad for the host.

Viruses also use reverse transcriptase to survive. Viruses called retroviruses have an RNA genome
and convert RNA back to DNA before hijacking the cell. There are several viruses that use reverse
transcriptase, such as Human T-lymphotropic virus (HTVL) type 1 and 2 and human
immunodeficiency virus (HIV). HTLV 1 may cause leukemia, a cancer of the white blood cells,
in some infected patients by mutating the white blood cell DNA. HIV is the most well known
example of retroviruses. When HIV infects a cell, it brings with it a genome made of RNA, not

28
DNA. The RNA enters the cell, and the reverse transcriptase copies the viral RNA back to DNA.
The viral DNA is cut and paste into the host cell DNA. The host cell gets tricked into making lots
of proteins for the virus. It doesn't notice the instructions have changed and keeps making lots of
new viruses! Although this is bad for the host, it is great for the virus, since the goal of a virus is
to survive and make more viruses.

Even though reverse transcriptase occurs in viruses, our cells have reverse transcriptase
enzymes that are helpful and even essential to our well-being! An enzyme called telomerase is an
important reverse transcriptase in our body. It helps to prevent our chromosomes from breaking
down over time and controls aging in cells

Inhibitors of DNA/RNA synthesis

 Some antibiotics prevent successful DNA replication in bacteria. A class of antimicrobials

called quinolones targets DNA gyrase, an important enzyme that helps unwind DNA for
replication. By removing gyrase from the equation, ciprofloxacin and similar antibiotics
effectively prevent the bacteria from multiplying.
 The rifamycins are a family of antibiotics that inhibit bacterial RNA polymerase.
Rifamycins work by binding to the bacterial DNA-dependent RNA polymerase, the
enzyme that is responsible for transcription of DNA into RNA. The antibiotic molecule is
thought to bind to the polymerase in such a way that it creates a wall that prevents the chain
of RNA from elongating.
 Quinolones and fluoroquinolones consists of drugs which act as chemotherapeutic agents
against bacteria. Quinolones and fluoroquinolones inhibit bacterial replication by blocking
their DNA replication pathway. During protein synthesis and DNA replication, the double-
stranded DNA needs to unwind into a single stranded structure, which allows for
complementary base pairing to occur and synthesis of mRNA to proceed. This unwinding
of DNA in the bacteria is done by enzymes in the bacteria called DNA gyrase or DNA
topoisomerase. Quinolones and fluoroquinolones inhibit this enzyme by binding to the A-
subunit of the enzyme due to which the bacteria is unable to replicate or even synthesize
proteins.

DNA Mutation

A Mutation occurs when a DNA gene is damaged or changed in such a way as to alter the genetic
message carried by that gene.

A Mutagen is an agent of substance that can bring about a permanent alteration to the physical
composition of a DNA gene such that the genetic message is changed.

Once the gene has been damaged or changed the mRNA transcribed from that gene will now carry
an altered message.

The polypeptide made by translating the altered mRNA will now contain a different sequence of
amino acids. The function of the protein made by folding this polypeptide will probably be changed
or lost.

29
A mutation, which may arise during replication and/or recombination, is a permanent change in
the nucleotide sequence of DNA. Damaged DNA can be mutated either by substitution, deletion
or insertion of base pairs. Mutations, for the most part, are harmless except when they lead to cell
death or tumor formation. Because of the lethal potential of DNA mutations cells have evolved
mechanisms for repairing damaged DNA.

Types of Mutations

There are three types of DNA Mutations: base substitutions, deletions and insertions.

1. Base Substitutions

Single base substitutions are called point mutations, recall the point mutation Glu -----> Val
which causes sickle-cell disease. Point mutations are the most common type of mutation and
there are two types.

 Transition: this occurs when a purine is substituted with another purine or when a
pyrimidine is substituted with another pyrimidine.
 Transversion: when a purine is substituted for a pyrimidine or a pyrimidine
replaces a purine.

Point mutations that occur in DNA sequences encoding proteins are either silent, missense or
nonsense.

30
 Silent: If abase substitution occurs in the third position of the codon there is a good chance
that a synonymous codon will be generated. Thus the amino acid sequence encoded by the
gene is not changed and the mutation is said to be silent.
 Missence: When base substitution results in the generation of a codon that specifies a
different amino acid and hence leads to a different polypeptide sequence. Depending on
the type of amino acid substitution the missense mutation is either conservative or
nonconservative. For example if the structure and properties of the substituted amino acid
are very similar to the original amino acid the mutation is said to be conservative and will
most likely have little effect on the resultant proteins structure / function. If the substitution
leads to an amino acid with very different structure and properties the mutation is
nonconservative and will probably be deleterious (bad) for the resultant proteins structure
/ function (i.e. the sickle cell point mutation).
 Nonsense: When a base substitution results in a stop codon ultimately truncating
translation and most likely leading to a nonfunctional protein.

31
2. Deletions

A deletion, resulting in a frameshift, results when one or more base pairs are lost from the DNA
(see Figure above). If one or two bases are deleted the translational frame is altered resulting in a
garbled message and nonfunctional product. A deletion of three or more bases leave the reading
frame intact. A deletion of one or more codons results in a protein missing one or more amino
acids. This may be deleterious or not.

3. Insertions

The insertion of additional base pairs may lead to frameshifts depending on whether or not
multiples of three base pairs are inserted. Combinations of insertions and deletions leading to a
variety of outcomes are also possible.

Mutagens

Chemical Mutagens change the sequence of bases in a DNA gene in a number of ways;

 mimic the correct nucleotide bases in a DNA molecule, but fail to base pair correctly
during DNA replication.
 remove parts of the nucleotide (such as the amino group on adenine), again causing
improper base pairing during DNA replication.
 add hydrocarbon groups to various nucleotides, also causing incorrect base pairing during
DNA replication.

Radiation High energy radiation from a radioactive material or from X-rays is absorbed by the
atoms in water molecules surrounding the DNA. This energy is transferred to the electrons which
then fly away from the atom. Left behind is a free radical, which is a highly dangerous and
highly reactive molecule that attacks the DNA molecule and alters it in many ways.
Radiation can also cause double strand breaks in the DNA molecule, which the cell's repair
mechanisms cannot put right.

Sunlight contains ultraviolet radiation (the component that causes a suntan) which, when
absorbed by the DNA causes a cross link to form between certain adjacent bases. In most normal
cases the cells can repair this damage, but unrepaired dimers of this sort cause the replicating
system to skip over the mistake leaving a gap, which is supposed to be filled in later.
Unprotected exposure to UV radiation by the human skin can cause serious damage and may
lead to skin cancer and extensive skin tumors.

Spontaneous mutations occur without exposure to any obvious mutagenic agent. Sometimes
DNA nucleotides shift without warning to a different chemical form (know as an isomer) which
in turn will form a different series of hydrogen bonds with it's partner. This leads to mistakes at
the time of DNA replication.

32
33