UNIT II

The process of gene expression simply refers to the events that transfer the information
content of the gene into the production of a functional product, usually a protein. Although there are
genes whose functional product is an RNA, including the genes encoding the ribosomal RNAs as well
as the transfer RNAs and certain other small RNAs, the vast majority of genes within the cell are
protein-encoding genes.

As shown in the figure above, the expressio n of a eukaryotic gene is a complex process involving a
variety of steps prior to the actual synthesis of a protein. These include the transcription of the gene
into the primary RNA product, processing of this initial gene transcript to remove intron sequences
and create the mature 3' terminus, transport of the
processed mRNA transcript to the cytoplasm, and then finally, translation of the messenger RNA into
protein. With very few exceptions, all of the genes that encode proteins follow this pathway.
What is a gene?
A gene is the basic physical and functional unit of heredity.
In 1909, Danish botanist Wilhem Johanssen coined the term “gene” as a physical and functional unit
of heredity.
Genes are made up of DNA.
Some genes act as instructions to make molecules called proteins. However, many genes do not code
for proteins. In humans, genes vary in size from a few hundred DNA bases to more than 2 million
bases.

Alleles are forms of the same gene with small differences in their sequence of DNA bases. These
small differences contribute to each person’s unique physical features. Every person has two copies
of each gene, one inherited from each parent.
The Genetic Code

Each amino acid is defined within the mRNA by a triplet of nucleotides called a codon. The
relationship between an mRNA codon and its corresponding amino acid is called the genetic code.

Translation of the mRNA template converts nucleotide-based genetic information into the “language”
of amino acids to create a protein product. A protein sequence consists of 20 commonly occurring
amino acids.

The three-nucleotide code means that there is a total of 64 possible combinations (43, with four
different nucleotides possible at each of the three different positions within the codon).

This number is greater than the number of amino acids and a given amino acid is encoded by more
than one codon (Fig.1). This redundancy in the genetic code is called degeneracy.

The first two positions in a codon are important for determining which amino acid will be
incorporated into a growing polypeptide, the third position, called the wobble position, is less critical.
In some cases, if the nucleotide in the third position is changed, the same amino acid is still
incorporated.

Out of the 64 possible triplets, 61 code for amino acids.

Three of the 64 codons do not code for an amino acid; they terminate protein synthesis, releasing the
polypeptide from the translation machinery. These are called stop codons or nonsense codons.

AUG, specifying the amino acid methionine, typically serves as the start codon to initiate translation.

The reading frame, the way nucleotides in mRNA are grouped into codons, for translation is set by
the AUG start codon near the 5’ end of the mRNA. Each set of three nucleotides following this start
codon is a codon in the mRNA message.

The genetic code is nearly universal. With a few exceptions, virtually all species use the same genetic
code for protein synthesis.

However, unusual amino acids such as selenocysteine and pyrrolysine have been observed in archaea
and bacteria. In the case of selenocysteine, the codon used is UGA (normally a stop codon).
Pyrrolysine uses a different stop codon, UAG. The incorporation of pyrrolysine requires the pylS gene
and a unique transfer RNA (tRNA) with a CUA anticodon.
Figure 1: This figure shows the genetic code for translating each nucleotide triplet in mRNA into an
amino acid or a termination signal in a nascent protein. The first letter of a codon is shown vertically
on the left, the second letter of a codon is shown horizontally across the top, and the third letter of a
codon is shown vertically on the right. (credit: modification of work by National Institutes of Health)

Figure 6: Comparison of translation in bacteria versus eukaryotes

The Protein Synthesis Machinery

In addition to the mRNA template, many molecules and macromolecules contribute to the process of
translation.

The composition of each component varies across taxa; for instance, ribosomes may consist of
different numbers of ribosomal RNAs (rRNAs) and polypeptides depending on the organism.
However, the general structures and functions of the protein synthesis machinery are comparable from
bacteria to human cells.

Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic
factors.

Ribosomes

A ribosome is a complex macromolecule composed of catalytic rRNAs (called ribozymes) and

structural rRNAs, as well as many distinct polypeptides. Mature rRNAs make up approximately 50%
of each ribosome.

Prokaryotes have 70S ribosomes, whereas eukaryotes have 80S ribosomes in the cytoplasm and rough
endoplasmic reticulum, and 70S ribosomes in mitochondria and chloroplasts.

Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and
reassociate during the initiation of translation.

In Prokaryotes like E. coli, the small subunit is described as 30S (which contains the 16S rRNA
subunit), and the large subunit is 50S (which contains the 5S and 23S rRNA subunits), for a total of
70S (Svedberg units are not additive).

Eukaryote ribosomes have a small 40S subunit (which contains the 18S rRNA subunit) and a large
60S subunit (which contains the 5S, 5.8S and 28S rRNA subunits), for a total of 80S.

The small subunit is responsible for binding the mRNA template, whereas the large subunit binds
tRNAs.

Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the
same direction: reading the mRNA from 5’ to 3’ and synthesizing the polypeptide from the N
terminus to the C terminus.

The complete structure containing an mRNA with multiple associated ribosomes is called a
polyribosome (or polysome).

In both bacteria and archaea, before transcriptional termination occurs, each protein- encoding
transcript is already being used to begin synthesis of numerous copies of the encoded polypeptide(s)
because the processes of transcription and translation can occur concurrently, forming polyribosomes
(Fig.2). The reason why transcription and translation can occur simultaneously is because both of
these processes occur in the same 5’ to 3’ direction, they both occur in the cytoplasm of the cell, and
because the RNA transcript is not processed once it is transcribed. This allows a prokaryotic cell to
respond to an environmental signal requiring new proteins very quickly.
In contrast, in eukaryotic cells, simultaneous transcription and translation is not possible. Although
polyribosomes also form in eukaryotes, they cannot do so until RNA synthesis is complete and the
RNA molecule has been modified and transported out of the nucleus.

Figure 2: In prokaryotes, multiple RNA polymerases can transcribe a single bacterial gene while
numerous ribosomes concurrently translate the mRNA transcripts into polypeptides. In this way, a
specific protein can rapidly reach a high concentration in the bacterial cell.

Transfer RNAs

Transfer RNAs (tRNAs) are structural RNA molecules and, depending on the species, many different
types of tRNAs exist in the cytoplasm.

Bacterial species typically have between 60 and 90 types. Serving as adaptors, each tRNA type binds
to a specific codon on the mRNA template and adds the corresponding amino acid to the polypeptide
chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the
language of proteins. As the adaptor molecules of translation, it is surprising that tRNAs can fit so
much specificity into such a small package. The tRNA molecule interacts with three factors:
aminoacyl tRNA synthetases, ribosomes, and mRNA.

Mature tRNAs take on a three-dimensional structure when complementary bases exposed in the
single-stranded RNA molecule hydrogen bond with each other (Fig.3). This shape positions the
amino-acid binding site, called the CCA amino acid binding end, which is a cytosine- cytosine-
adenine sequence at the 3’ end of the tRNA, and the anticodon at the other end. The anticodon is a
three-nucleotide sequence that bonds with an mRNA codon through complementary base pairing.

An amino acid is added to the end of a tRNA molecule through the process of tRNA “charging,” during
which each tRNA molecule is linked to its correct or cognate amino acid by a group of enzymes
called aminoacyl tRNA synthetases. At least one type of aminoacyl tRNA synthetase exists for each
of the 20 amino acids. During this process, the amino acid is first activated by the addition of
adenosine monophosphate (AMP) and then transferred to the tRNA, making it a charged tRNA, and
AMP is released.
Figure 3: (a) After folding caused by intramolecular base pairing, a tRNA molecule has one end that
contains the anticodon, which interacts with the mRNA codon, and the CCA amino acid binding end.
(b) A space-filling model is helpful for visualizing the three-dimensional shape of tRNA. (c) Simplified
models are useful when drawing complex processes such as protein synthesis.

Exercise
1. Describe the structure and composition of the prokaryotic ribosome.
2. In what direction is the mRNA template read?
3. Describe the structure and function of a tRNA.
Prokaryotic Genome: Chromosomal & Plasmid

Prokaryotic cells do not contain nuclei or other membrane-bound organelles. The word "prokaryote"
literally means "before the nucleus." The nucleoid is simply the area of a prokaryotic cell in which the
chromosomal DNA is located.

In prokaryotes, the genome is composed of a single, double-stranded DNA molecule in the form of a
loop or circle. The region in the cell containing this genetic material is called a nucleoid.
Some prokaryotes also have smaller loops of DNA called plasmids that are not essential for normal
growth.

A. Chromosomal DNA
DNA Supercoiling

 Prokaryotes compress their DNA into smaller spaces is through supercoiling (Figure 1). The
original coils fold over one another and form a condensed ball. When this type of twisting
happens to a bacterial genome, it is known as supercoiling.
 Genomes can be negatively supercoiled, meaning that the DNA is twisted in the opposite
direction of the double helix, or positively supercoiled, meaning that the DNA is twisted in
the same direction as the double helix. Most bacterial genomes are negatively supercoiled
during normal growth.
Proteins Involved in Supercoiling

During the 1980s and 1990s, researchers discovered that multiple proteins act together to fold and
condense prokaryotic DNA. In particular, one protein called HU, which is the most abundant protein
in the nucleoid, works with an enzyme called topoisomerase I to bind DNA and introduce sharp bends
in the chromosome, generating the tension necessary for negative supercoiling. Recent studies have
also shown that other proteins, including integration host factor (IHF), can bind to specific
sequences within the genome and introduce additional bends. The folded DNA is then organized into
a variety of conformations that are supercoiled and wound around tetramers of the HU protein,
much like eukaryotic chromosomes are wrapped around histones.
Once the prokaryotic genome has been condensed, DNA topoisomerase I, DNA gyrase, and other
proteins help maintain the supercoils. One of these maintenance proteins, H-NS, plays an active
role in transcription by modulating the expression of the genes involved in the response to
environmental stimuli. Another maintenance protein, factor for inversion stimulation (FIS), is
abundant during exponential growth and regulates the expression of more than 231 genes, including
DNA topoisomerase I.

Accessing Supercoiled Genes

Supercoiling explains how chromosomes fit into a small corner of the cell, but how do the proteins
involved in replication and transcription access the thousands of genes in prokaryotic chromosomes
when everything is packaged together so tightly? It has been determined that prokaryotic DNA
replication occurs at a rate of 1,000 nucleotides per second, and prokaryotic transcription occurs at a
rate of about 40 nucleotides per second, so bacteria must have highly efficient methods of accessing
their DNA strands. But how?
Researchers have noted that the nucleoid usually appears as an irregularly shaped mass within the
prokaryotic cell, but it becomes spherical when the cell is treated with chemicals to inhibit
transcription or translation. Moreover, during transcription, small regions of the chromosome can be
seen to project from the nucleoid into the cytoplasm (i.e., the interior of the cell), where they unwind
and associate with ribosomes, thus allowing easy access by various transcriptional proteins. These
projections are thought to explain the mysterious shape of nucleoids during active growth. When
transcription is inhibited, however, the projections retreat into the nucleoid, forming the
aforementioned spherical shape.
Because there is no nuclear membrane to separate prokaryotic DNA from the ribosomes within the
cytoplasm, transcription and translation occur simultaneously in these organisms. This is strikingly
different from eukaryotic chromosomes, which are confined to the membrane-bound nucleus
 during most of the cell cycle. In eukaryotes, transcription must be completed in the nucleus before the
newly synthesized mRNA molecules can be transported to the cytoplasm to undergo translation into
proteins.
Operons
Furthermore, unlike eukaryotic chromosomes, most prokaryotic genomes are organized into
polycistronic operons, or clusters of more than one coding region attached to a single
promoter, separated by only a few base pairs. The proteins encoded by each operon often collaborate
on a single task, such as the metabolism of a sugar into by-products that can be used for energy
(Figure 3).

Figure 3: The prokaryotic lac operon.

Three structural genes code for proteins involved in lactose import and metabolism
in bacteria. The genes are organized together in a cluster called the lac operon.

B. Plasmid DNA

Prokaryotic cells often contain one or more plasmids (i.e., extrachromosomal DNA molecules that are
either linear or circular). These pieces of DNA differ from chromosomes in that they are typically
smaller and encode nonessential genes, such as those that aid growth in specific conditions or encode
antibiotic resistance. Borrelia, for instance, contains more than 20 circular and linear plasmids that
encode genes responsible for infecting ticks and humans. Plasmids are often much smaller than
chromosomes (i.e., less than 1,500 kilobases), and they replicate independently of the rest of the
genome. However, some plasmids are capable of integrating into chromosomes or moving from cell
to cell.

Bacteria can exchange these plasmids with other bacteria, sometimes receiving beneficial new genes
that the recipient can add to their chromosomal DNA. Antibiotic resistance is one trait that often
spreads through a bacterial colony through plasmid exchange.

Differences between Prokaryotic & Eukaryotic Chromosomes

The organization of prokaryotic DNA therefore differs from that of eukaryotes in several important
ways. The most notable difference is the condensation process that prokaryotic DNA molecules
undergo in order to fit inside relatively small cells. Other differences, while not as dramatic, are
summarized in Table 1.
 Prokaryotic Chromosomes Eukaryotic Chromosomes

 Many prokaryotes contain a single  Eukaryotes contain multiple linear

circular chromosome. chromosomes.
 Prokaryotic chromosomes are  Eukaryotic chromosomes are
condensed in the nucleoid via DNA condensed in a membrane-bound
supercoiling and the binding of nucleus via histones.
various architectural proteins.  In eukaryotes, transcription occurs in
 Because prokaryotic DNA can the nucleus, and translation occurs in
interact with the cytoplasm, the cytoplasm.
transcription and translation occur  Most eukaryotes contain two copies
simultaneously. of each gene (i.e., they are diploid).
 Most prokaryotes contain only one  Some eukaryotic genomes are
copy of each gene (i.e., they are organized into operons, but most are
haploid). not.
 Nonessential prokaryotic genes are  Extrachromosomal plasmids are not
commonly encoded on commonly present in eukaryotes.
extrachromosomal plasmids.  Eukaryotes contain large amounts of
 Prokaryotic genomes are efficient noncoding and repetitive DNA.
and compact, containing little
repetitive DNA.

Note: Additional study

Vibrio cholerae, the bacteria that causes cholera, contains two circular chromosomes. Furthermore,
the DNA molecules of Archaea, a single-celled, nonbacterial prokaryotes that share many similarities
with eukaryotes, can be negatively supercoiled, positively supercoiled, or not supercoiled at all. It is
important to note that archaeans are the only group of prokaryotes that use eukaryote-like histones,
rather than the architectural proteins described above, to condense their DNA molecules.

Other DNA Differences Between Prokaryotes and Eukaryotes

Most prokaryotes reproduce asexually and are haploid, meaning that only a single copy of each
gene is present. This makes it relatively easy to generate mutations in the lab and study the resulting
phenotypes. By contrast, eukaryotes that reproduce sexually generally contain multiple chromosomes
and are said to be diploid, because two copies of each gene exist—with one copy coming from each of
an organism's parents.
Yet another difference between prokaryotes and eukaryotes is perhaps due to the space constraints of
packing so many essential genes onto a single chromosome, prokaryotes can be highly efficient in
terms of genomic organization. Very little space is left between prokaryotic genes. As a result,
noncoding sequences account for an average of 12% of the prokaryotic genome, as opposed to
upwards of 98% of the genetic material in eukaryotes.
Organisation of the Eukaryotic Genome

a. Chromosomal Genome

Eukaryotic nuclear genomes characteristics:

o Eukaryotic genomes possess two unique characteristics that pose a significant challenge in
terms of information processing.
1. The typical multicellular eukaryotic cell has a significantly larger genome than a
prokaryotic cell.
2. Due to cell specialisation, many genes can only be expressed in certain types of cells.
o The human genome, with its estimated 35,000 genes, contains a large amount of DNA that
does not direct the synthesis of RNA or protein.
o The organisation of eukaryotic DNA is complex. The DNA-protein complex known as
chromatin is not only associated with proteins but is also structured at a higher level than the
DNA-protein complex in prokaryotes.
o Eukaryotic cells possess a significantly higher concentration of DNA in their nuclei compared
to prokaryotic cells.
The Structure of Chromatin

Chromatin is the complex structure of DNA and protein that makes up chromosomes and consists of
linear unbroken double-stranded DNA. There are two types of chromatin:

1. Euchromatin
2. Heterochromatin

Euchromatin: This is a lightly packed form of chromatin that is rich in genes and often under active
transcription (although not always). Euchromatin contrasts sharply with heterochromatin, which is
densely packed and much less available for transcription. The euchromatic region constitutes 92% of
the human genome.

Heterochromatin: This is a densely compacted form of DNA, or compressed DNA, which comes in
various variants. These variants range between facultative heterochromatin and constitutive
heterochromatin. Both play a role in how genes are expressed.

The main proteins that make up chromatin are called histones and DNA is wrapped around these
histone proteins. There are five main types of histones associated with the eukaryotic genome: H1,
H2A, H2B, H3 & H4. These basic proteins are positively charged at normal pH levels, making it easy
for negatively charged DNA to bind to them.

How DNA is Packed

Level 1: Histone Proteins

o Histone proteins are present at the first level.

o The negatively charged DNA forms a strong bond with the positively charged amino acids.
o All eukaryotes contain the five main types of histones.
o The nucleosome resembles beads on a string in unfolded chromatin.
o The beaded thread appears to remain largely unchanged during the cell cycle.
o Histones temporarily detach from the DNA during DNA replication. They stay with the DNA
during transcription.
o Nucleosomes allow RNA-synthesising polymerases to move along the DNA by changing
their size and position.

Level 2: As chromosomes undergo mitosis, the beaded thread coils into the 30-nm chromatin fibre.

Level 3: This fibre forms looped domains that are connected to a nonhistone protein scaffold.

Level 4: The metaphase chromosome is created by the coiling and folding of the looping domains.

Interphase chromatin is generally less compressed than mitosis chromatin, with the 30-nm fibres and
looped domains remaining unaffected. Each chromosome's chromatin occupies a limited area within
the interphase nucleus. Chromosomes in interphase have heterochromatin, which is highly condensed,
and euchromatin, which is less condensed.

DNA-Level Organisation of the Eukaryotic Genome

The majority of DNA in eukaryotes (97% in humans) cannot code for protein or RNA.

1. Regulatory sequences are found in non-coding regions.

2. They are typically introns.
3. The genome contains many copies of repetitive DNA.

In mammals, tandemly repetitive DNA, also known as satellite DNA, accounts for 10-15% of the
genome. These areas are denser than the surrounding areas, and differential ultracentrifugation results
in the formation of a distinct band around them. The total length of DNA at each site distinguishes
between three different types of satellite DNA.

Note: Additional study

Some genetic disorders are caused by unusually long sections of tandemly repeated nucleotide triplets
within the affected gene.

o Fragile X syndrome, for example, is primarily caused by numerous CGG repeats in the fragile
X gene.
o Huntington’s disease is caused by the repetition of CAG, which is translated into proteins
with a long string of glutamines.
o The number of repeats is linked with the severity of the disease and the age at which these
disorders first appear.

Most mammalian genomes contain interspersed repetitive DNA to a degree of 25–40%.

These repetitive sequences appear throughout the genome at various locations. They are similar but
usually not identical to each other.

b. Organellar Genome

In eukaryotes, DNA and genes also exist outside of the chromosomes found in the nucleus. Both the
chloroplast and mitochondrion have circular chromosomes (Figure 2). These organellar genomes
are often present in multiple copies within each organelle. In most sexually reproducing species,
organellar chromosomes are inherited from only one parent, usually the one that produces the largest
gamete. Thus, in mammals, angiosperms, and many other organisms, mitochondria and chloroplasts
are inherited only through the mother (maternally).

These organelles are likely the remnants of prokaryotic endosymbionts that entered the cytoplasm of
ancient progenitors of today’s eukaryotes (endosymbiont theory). These endosymbionts had their
own, circular chromosomes, like most bacteria that exist today. Chloroplasts and mitochondria
typically have circular chromosomes that behave more like bacterial chromosomes than eukaryotic
chromosomes, i.e. these organellar genomes do not undergo mitosis or meiosis.

The mitochondrial chromosome also encodes various tRNAs and rRNAs used in translation of the
genes encoded on this chromosome. Other proteins required by the mitochondrion are encoded in the
nuclear genome, and are translated in the cytoplasm and imported into the organelle. Circular
organellar chromosomes such one as this are typical of almost all eukaryotes. (From Rogaev et al,
2006). Recent (Rohland et al, 2010) mtDNA work indicates that mammoths are more closely related
to Indian elephants than to either of the African species.
Flow of Genetic Information

In bacteria, archaea, and eukaryotes, the primary role of DNA is to store heritable information that
encodes the instruction set required for creating the organism in question. While we have gotten much
better at quickly reading the chemical composition (the sequence of nucleotides in a genome and
some of the chemical modifications that are made to it), we still don't know how to reliably decode all
of the information within and all of the mechanisms by which it is read and ultimately
expressed.

There are, however, some core principles and mechanisms associated with the reading and
expression of the genetic code whose basic steps are understood and that need to be part of the
conceptual toolkit for all biologists. Two of these processes are transcription and translation, which
are the coping of parts of the genetic code written in DNA into molecules of the related polymer
RNA and the reading and encoding of the RNA code into
proteins, respectively.

Followed by the process of creating a variety of functional RNA molecules (that may have various
structural, catalytic or regulatory roles) including so called messenger RNA (mRNA) molecules that
carry the information required to synthesize proteins. Likewise, we focus on challenges and questions
associated with the process of translation, the process by which the ribosomes synthesize proteins.

The basic flow of genetic information in biological systems is often depicted in a scheme known as
"the central dogma". This scheme states that information encoded in DNA flows into RNA via
transcription and ultimately to proteins via translation. Processes like reverse transcription (the
creation of DNA from and RNA template) and replication also represent mechanisms for propagating
information in different forms. This scheme, however, doesn't say anything per se about how
information is encoded or about the mechanisms by which regulatory signals move between the
various layers of molecule types depicted in the model.

Figure 1. The flow of genetic information.

Attribution: Marc T. Facciotti (original
work)
Fine Structure of Gene: Cistron, muton and recon

In order to define the units of function, recombination and mutation, Benzer, coined the terms
cistron (unit of function), recon (unit of recombination) and muton (unit of mutation). Cistron was
defined as a unit, the elements (alleles) of which exhibit cis- trans phenomenon.

Gene, is a unit of heredity.

The gene may be subdivided into different units according to Benzer such as Recon, Muton, Cistron
and Operon.
Cistron
It is the functional unit which can synthesize one polypeptide.
It is the segment of DNA that contains all the information necessary for the production of a single
polypeptide and includes both the coding sequences and regulatory sequences that are transcription
start and stop signals. The alleles exhibit the cis-trans phenomenon.
cistron is not a synonym for gene.

Recon
It is that smallest portion of a gene which can undergo crossing over and recombination
and may be as small as a single nuclecotide pair.
Recon is a unit of recombination. This term was coined by Seymour Benzer for the smallest
recombinant unit. Recon is a part or segment of the present cistron sequence. It is a region of a
gene in which there can be no crossing-over, now known to be a single nucleotide pair.

Muton
It is the smallest unit of a gene that can undergo mutation and can involve a pair of nucleotides.
It describes the smallest mutable site within a cistron. It is the smallest part of a gene that can be
involved in a mutation event. It is mostly to be a single nucleotide pair.
Sometimes a muton can be a normal nucleotide or it might be a radioactive element and sometimes
muton might consist of 3 to 4 nucleotides.

Operon
It is a group of genes having an operator a structural gene and other genes in sequence
which all function as a unit.

Exons and Introns

In Eukaryotes, the genes on the DNA strand have coding regions called exons interrupted by non-
coding DNA segments which do not carry genetic information called introns. This led to the
concept of interrupted genes or discontinuous genes. Such genes while producing m-RNA will first
form a primary transcript which will then cut off the introns to form the functional m-RNA and
this is called splicing.
 DNA REPLICATION

DNA replication, also known as semi-conservative replication, is the process by which DNA is
doubled. This is an important process taking place within the dividing cell.

 the structure of DNA,

 the steps involved in DNA replication (initiation, elongation and termination) and
 the clinical consequences that can occur when this process goes wrong.

The DNA (Deoxyribonucleic acid)

 DNA is the genetic material in most of the organisms except some viruses, which have
an RNA genome, e.g. TMV (Tobacco mosaic virus)
 RNA mostly acts as a messenger, an adapter and has a catalytic function
 Number of base pairs (bp) or nucleotides determines the length of the DNA
Human DNA (haploid) – 3.3 x 109 bp Bacteriophage 𝜙 x 174 –
5386 nucleotides Bacteriophage 𝝀 – 48502 bp

E. coli – 4.6 x 106 bp

DNA Structure

DNA is made up of millions of nucleotides. These are molecules composed of a deoxyribose sugar,
with a phosphate and a base (or nucleobase) attached to it. These nucleotides are attached to each
other in strands via phosphodiester bonds to form a ‘sugar-phosphate backbone’. The bond formed is
between the third carbon atom on the deoxyribose sugar of one nucleotide (known as the 3’) and the
fifth carbon atom of another sugar on the next nucleotide (known as the 5’).

N.B: 3′ is pronounced ‘three prime’ and 5′ is pronounced ‘five prime’.

There are two strands of DNA, which run in opposite (antiparallel) directions to each other. These
strands are attached to each other throughout their lengths via the bases on each nucleotide.

There are 4 different bases associated with DNA: Cytosine, Guanine, Adenine, and Thymine. In
normal DNA strands, cytosine binds to guanine, and adenine binds to thymine. When bound together,
the two strands form a double helix structure.
Stages of DNA replication Fig 1 – The Structure of RNA and DNA

Schematic of Watson and Crick's basic model of DNA replication.

1. DNA double helix.
2. Hydrogen bonds break and helix opens.
3. Each strand of DNA acts as a template for synthesis of a new, complementary strand.
4. Replication produces two identical DNA double helices, each with one new and one old
strand.
 DNA replication can be thought of in three stages:

elongation and termination

Initiation

DNA synthesis is initiated at particular points within the DNA strand known as ‘origins’, which have
specific coding regions. These origins are targeted by initiator proteins, which go on to recruit more
proteins that help aid the replication process, forming a replication complex around the DNA origin.
Multiple origin sites exist within the DNA’s structure; when replication of DNA begins, these
sites are referred to as replication forks.

Within the replication complex is the DNA helicase. This enzyme unwinds the double helix and
exposes each of the two strands so that they can be used as a template for replication. It does this by
hydrolysing the ATP used to form the bonds between the nucleobases, thereby breaking the bond
holding the two strands together.

DNA primase is another enzyme that is important in DNA replication. It synthesises a small RNA
primer, which acts as a ‘kick-starter’ for DNA polymerase. This enzyme is ultimately responsible
for the creation and expansion of new strands of DNA.

Elongation

Once DNA Polymerase has attached to the two unzipped strands of DNA (i.e. the template
strands), it is able to start synthesising new strands of DNA to match the templates. DNA polymerase
is only able to extend the primer by adding free nucleotides to the 3’ end.

One of the template strands is read in a 3’ to 5’ direction, therefore the new strand will be formed in a
5’ to 3’ direction. This newly formed strand is referred to as the leading
strand. Along the leading strand, DNA primase only needs to synthesise an RNA primer once, at the
beginning, to initiate DNA polymerase. This is because DNA polymerase is able to extend the new
DNA strand by reading the template 3′ to 5′, synthesising in a 5′ to 3′ direction as noted above.

However, the other template strand (the lagging strand) is antiparallel and is therefore read in a 5’ to
3’ direction. Continuous DNA synthesis, as in the leading strand, would need to be in the 3′ to 5′
direction, which is impossible as DNA polymerase cannot add bases to the 5′ end. Instead, as the
helix unwinds, RNA primers are added to the newly exposed bases on the lagging strand and DNA
synthesis occurs in fragments, but still in the 5′ to 3′ direction as before. These fragments are known
as Okazaki fragments.

Termination

The process of expanding the new DNA strands continues until there is either no more DNA template
strand left to replicate (i.e. at the end of the chromosome) or two replication forks meet and
subsequently terminate. The meeting of two replication forks is not regulated and happens randomly
along the course of the chromosome.

Once DNA synthesis has finished, the newly synthesised strands are bound and stabilised. For the
lagging strand, two enzymes are needed to achieve this stabilisation: RNAase H removes the RNA
primer at the beginning of each Okazaki fragment, and DNA ligase joins these fragments together to
create one complete strand.

Fig 2 – Diagrammatic representation of DNA replication

single base substitutions can result in a ‘silent mutation‘ in which the overall gene is not affected,
however, in diseases such as sickle cell anaemia, it results in the strand coding for a different protein.

In this case, an adenine base is swapped for a thymine base in one of the genes coding for
haemoglobin; this results in glutamic acid being replaced by valine. When this is being transcribed
into a polypeptide chain, the properties it possesses are radically changed, as glutamic acid is
hydrophilic, whereas valine is hydrophobic. This hydrophobic region results in haemoglobin having
an abnormal structure that can cause blockages of capillaries, leading to ischaemia and potentially
necrosis of tissues and organs – this is known as a vaso-occlusive crisis.

These crises are typically managed with analgesia, including opioids and NSAIDs depending on the
severity. Red blood cell transfusions may be required in emergencies, for example, if the blockage
occurs in the lungs.

By The National Heart, Lung, and Blood Institute (NHLBI) [Public domain], v ia Wikimedia
Commons

Fig 3 – The difference in structure between normal red blood cells and those affected by sickle cell
disease.

DNA is the cell’s genetic material, passed down to daughter cells. As a result, the entire DNA
molecule should be replicated before cell division. As a result, DNA replication is a fundamental
process in all organisms, whether prokaryotic or eukaryotic. Using the current DNA molecule as a
template, the DNA replication process generates two identical copies of daughter DNA molecules.
The replication mechanism in eukaryotic and prokaryotic species is very similar, with few variances.
The differences in replication between prokaryotic and eukaryotic organisms are primarily due to
variations in genomic size and complexity. The eukaryotic cell has much more DNA than the
prokaryotic cell, which is tightly packed as a chromosome in the eukaryotic cell’s nucleus. For
example, the human genome is about 1000 times bigger than the E. coli genome.
Eukaryotic and Prokaryotic Replication: Similarities
1. Before entering the nuclear division, both eukaryotic and prokaryotic DNA replications
occur.
2. Double-stranded DNA is used in both bacterial and eukaryotic replication.
3. DNA helicase is responsible for the unravelling of both eukaryotic and prokaryotic DNA.
4. Single-wrecked DNA-binding protein stabilizes the unwinding DNA swrecks(SSB).
5. DNA replication is a multistep process carried out by an enzyme complex known as DNA
polymerases in prokaryotic and eukaryotic cells.

Eukaryotic and Prokaryotic Replication: Differences

Prokaryotic Replication
Prokaryotic replication is when prokaryotes are archaea and bacteria repeat their genome to create a
copy that may be turned into a daughter. In their cytoplasm, prokaryotes have a double-stranded
circular molecule. Prokaryotic DNA has a single replication origin. By disrupting the hydride bonds
between the nitrogen bases, helicase unwinds at the replication site. The replication fork is the
resulting Y shaped structure. Because prokaryotic DNA has a single replication origin, only 2
replication forks are generated throughout the cloning process. These two cloning forks work in both
directions.
Single strand DNA binding protein stabilizes the two unwinding strands that act as replication
template strands. RNA primase is an enzyme that produces a 5-10 base pair prolonged RNA basal
complementary to the template strand.

Eukaryotic Replication
The process by which the eukaryote replicates before cell division is known as eukaryotic DNA
replication. Though the underlying process of eukaryotic replication is identical to that of prokaryotic
replication, there are notable changes owing to eukaryotic DNA’s size and structure. DNA in
Eukaryotes is made up of double wrecked linear molecules. The quantity of eukaryotic DNA is about
fifty times more than prokaryotic DNA. Furthermore, eukaryotic DNA is densely filled
with histones within the cell’s nucleus. As a result, DNA replication happens in three
elongate, and terminate.

Eukaryotic and Prokaryotic Replication: Differences

Prokaryotic Replication Eukaryotic Replication

Prokaryotic DNA replication takes place in the The replication occurs in the nucleus of cell
cytoplasm of cell

Each DNA molecule has a single place of origin A single DNA molecule has several sites of origin

The replication origin is made up of 100-200 Each replication origin has around 150 nucleotides
nucleotides or more

The replication process is bidirectional, and only In multiple replication bubbles, many replication
two replication forks are created forks develop

There is just one replicon on the bacterial There are approximately 50,000 replicons on the
chromosome eukaryotic chromosome

Polymerase I and III carry out the procedure DNA Polymerase is responsible for the process, and
DNA gyrase is necessary. It is not required to use
DNA gyrase

The pieces of Okazaki are huge. They range in The Okazaki fragments are tiny, measuring
length from 1000 to 2000 nucleotides between 100 and 200 nucleotides in length

It is a fast process that adds over 2000 It is a time-consuming process, with around a
nucleotides per second hundred nucleotides inserted every second

The DNA is double-stranded and circular The DNA is double-stranded and linear

Conclusion
DNA replication in eukaryotic and prokaryotic cells is involved in genome duplication before cell
division. Both bacterial and eukaryotic replication mechanisms are comparable. However, eukaryotic
replication is a more tough process due to the eukaryotic genome’s size and complexity. As a result,
eukaryotic DNA replication takes place by developing numerous replication origins. On the other
hand, prokaryotic replication proceeds through a single cloning origin. However, because prokaryotic
replication occurs quickly, both replication processes require the same amount of time. As a result, the
primary distinction between eukaryotic and prokaryotic replication is based on the complexity and
size of each kind of genome.

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.
Replisome/replicon:

1. Individual units of replication are called replicons, each of which contains a replication origin .

2. Replisome is composed of many proteins, which are required for replication such
as DNA polymerase. helicase, primase, ligase, topoisomerase, etc
3. Replication starts at the origin and continues until the entire replicon has been replicated.
4. Bacterial chromosomes have a single replication origin, whereas eukaryotic
chromosomes contain many.
5. The unwinding of the double helix generates a loop, termed a replication bubble .
6. The point of unwinding, where the two single nucleotide strands separate from the double-
stranded DNA helix, is called a replication fork
7. If there are two replication forks, one at each end of the replication bubble, the forks
proceed outward in both directions in a process called bidirectional
replication.
3A_Mechanisms_of_Microbial_Genetics/9.4%3A_Protein_Synthesis_(Translation)
 DNA polymerases

DNA polymerase 𝝳 is the main enzyme for replication.

DNA polymerase III is the main enzyme responsible for replication in prokaryotes. In eukaryotes,

DNA polymerase I removes the RNA primer by 5’→3’ exonuclease activity

and replaces the primer by its polymerase activity in the lagging strand.

Repair

The replication process is a humongous task and it is important to maintain the integrity of the
genome. Apart from replication errors, DNA repair is the continuous process to rectify any errors
in the genome due to DNA damage. There are various mechanisms by which DNA is repaired.

Proofreading

DNA replication is not perfect and there occurs an error after every 10 4 to 105 nucleotides added.
Removing the incorrect nucleotide sequence or mismatched nucleotides from the newly
synthesised strand is very important for the functionality of proteins, which can even lead to
cancer. DNA polymerases remove incorrect pairs by exonuclease activity. They move one step
back and remove the mismatched pair by 3’→5’ exonuclease activity. This is known as
proofreading.

DNA polymerases are also involved in the post-replication DNA repair processes and also in
translesion synthesis by which DNA polymerase copies unrepaired part of the DNA blocking the
progression of replication.

DNA Polymerase Structure and Types

The structure of most of the DNA polymerases resembles a hand, which is holding active sites. The
active site of the enzyme has two parts. At the insertion site, nucleotides are added. After adding, the
newly formed base- pair migrates to the post-insertion site.
 Prokaryotic DNA Polymerase Types and Function
There are five DNA polymerases identified in E.coli. All the DNA polymerases differ in structure,
functions and rate of polymerization and processivity.

DNA Polymerase I is coded by polA gene. It is a single polypeptide and has a role in recombination
and repair. It has both 5’→3’ and 3’→5’ exonuclease activity. DNA polymerase Ⅰ removes the RNA
primer from lagging strand by 5’→3’ exonuclease activity and also fills the gap.

DNA Polymerase II is coded by polB gene. It is made up of 7 subunits. Its main role is in repair and
also a backup of DNA polymerase III. It has 3’→5’ exonuclease activity.

DNA Polymerase III is the main enzyme for replication in E.coli. It is coded by polC gene. The
polymerization and processivity rate is maximum in DNA polymerase III. It also has proofreading
3’→5’ exonuclease activity.

DNA polymerase III of E.coli is made up of a total of 13 subunits, which comprises 9 different types
of subunits.

 It consists of two core domains made up of 𝜶, 𝟄, and 𝞱 subunits. It is attached to the 𝝲

𝞽2𝝲𝝳𝝳’. Additional subunits 𝟀 and 𝟁 are attached to the clamp-loading

complex or clamp-loading complex, which is made up of five subunits,
 complex. 𝞫 subunits make two clamps with a dimer each. They increase the processivity of
the DNA polymerase III.
DNA Polymerase IV is coded by dinB gene. Its main role is in DNA repair during SOS response,
when DNA replication is stalled at the replication fork. DNA polymerase II, IV and V are translesion
polymerases.

DNA Polymerase V is also involved in translesion synthesis during SOS response and DNA
repair. It is made up of UmuC monomer and UmuD dimer.
 Eukaryotic DNA Polymerase Types and Function

Like prokaryotic cells, eukaryotic cells also have many DNA polymerases, which perform different

replication is mainly done by DNA polymerase 𝝳 and 𝜶. There are at least 15 DNA polymerases
functions, e.g. mitochondrial DNA replication, nuclear DNA replication, etc. The nuclear DNA

identified in human beings.

 DNA polymerase 𝝳 – It is the main enzyme for replication in eukaryotes. It also has 3’→5’

DNA polymerase 𝜶 – The main function of DNA polymerase 𝜶 is to synthesize primers.

exonuclease activity for proofreading.

The smaller subunit has a primase activity. The largest subunit has polymerization

polymerase 𝝳.
activity. It forms a primer for Okazaki fragments, which is then extended by DNA

 DNA polymerase 𝟄 – The main function is DNA repair. It removes primers for Okazaki

DNA polymerase 𝝲 – It is the main replicative enzyme for mitochondrial DNA.

fragments from the lagging strand.

 How does DNA Polymerase work?

and attacks the incoming deoxyribonucleoside triphosphate at the 𝜶-phosphorus, leading to

The reaction is phosphoryl group transfer. The 3’-OH group of the growing strand acts as a nucleophile

phosphodiester bond formation. Inorganic phosphate is released in the reaction.

(dNMP)n + dNTP → (dNMP)n+1 + PPi

All the DNA polymerases require two Mg ions at the active site. It is important to note that DNA
polymerase can only add nucleotides at the 3′ end of the growing strand, that is why replication
always occurs in the 5’→3’ direction. They cannot initiate the formation of new DNA.

They need a template strand, which guides the polymerisation reaction. They also need a primer for
their action as they can only add nucleotides at 3’ OH group. The primer can be a short segment
of RNA, DNA or both.
Generally, the primer is an RNA oligonucleotide in the living system.

After adding a nucleotide, the DNA polymerase can either dissociate or move along to add more
nucleotides. It depends on the processivity of DNA polymerase and it differs in different DNA
polymerases.
Replication is a highly accurate process and even the change in a single nucleotide can cause
mutation. To avoid this there are two mechanisms by which DNA polymerases ensure that
there are no discrepancies.

 The geometry of the active sites allows only the correct nucleotide base pairs to fit. But this
is not sufficient and it is seen that it can add an incorrect nucleotide after correctly adding
104 to 105 nucleotides.
 To correct this type of errors, DNA polymerase has 3’→5’ exonuclease activity. DNA
polymerase checks each of the added nucleotides and removes the nucleotide if there is a
mismatch. This is known as proofreading. In DNA polymerase I, there are different active
sites for polymerizing and proofreading functions.

Table 14.5.114.5.1: Difference between Prokaryotic and Eukaryotic Replication

Property Prokaryotes Eukaryotes

Origin of replication Single Multiple

Rate of replication 1000 nucleotides/s 50 to 100 nucleotides/s

DNA polymerase types 5 14

Telomerase Not present Present

RNA primer removal DNA pol I RNase H

Strand elongation DNA pol III Pol δ, pol ε

Sliding clamp Sliding clamp PCNA

Q1

What is the role of DNA polymerase?

The main function of DNA polymerase is to synthesize DNA by the process of replication. It adds
deoxyribonucleotides at the 3′-OH group of the growing DNA strand and synthesises the new strand in 5’→3’
direction.
Also see: Nucleotide
Different DNA polymerases perform specific functions. In prokaryotes, DNA polymerase III is the main

primer and filling the gaps. In eukaryotes, DNA polymerase 𝝳 is the main enzyme for replication. Other
enzyme responsible for replication. DNA polymerase I and II have a role to play in repair, removing the

DNA polymerases are involved in the repair, proofreading and primer removal.
Q2

What are the 3 main functions of DNA polymerase?

The main function of DNA polymerase is to replicate and form new DNA strands and repair any
mismatch or damage in the DNA. DNA polymerase duplicates the cellular DNA content every time a
cell divides so that there is an equal distribution of DNA to the daughter cells. The three main functions of
DNA polymerase are:

 5’→3’ polymerisation – it is required for replication and to add nucleotides at the

3’- OH group of the growing DNA strand and filling the gaps.
 3’→5’ exonuclease – it is required for proofreading and DNA polymerase removes
any incorrectly added nucleotides while replication.
 5’→3’ exonuclease – It is responsible for removing RNA primers and repair.

Q3
What are the types of DNA polymerase?
There are various different types of DNA polymerase identified in prokaryotes
and eukaryotes:

 Prokaryotes contain five different DNA polymerases named from I to V.

DNA polymerase III – is the main enzyme responsible for replication. Other DNA
polymerases take part in repair, removing, primer, proofreading, translesion
synthesis.

DNA polymerase 𝝳 and 𝜶 – The main DNA polymerases for nuclear

 Eukaryotes also contain many different types of DNA polymerase.

replication. DNA polymerase 𝝲 – It is involved in mitochondrial DNA

replication.
 DNA polymerase 𝟄 – Its main function is to repair DNA. It removes primer from
Okazaki fragments.

Q4

DNA polymerase III is used in the replication process in prokaryotic cells and DNA polymerase 𝝳 is the main
Which DNA polymerase is used in DNA replication?

enzyme for replication in eukaryotic cells.

Q5

What’s the difference between DNA polymerase and RNA polymerase?

DNA polymerase synthesises DNA during replication and RNA polymerase synthesises RNA
during transcription.
Q6

Does DNA polymerase need a primer?

Yes, DNA polymerase requires a primer as they can add a nucleotide to the 3’-OH group of a DNA
strand. DNA polymerases cannot initiate the replication process and they need a primer to add
nucleotides.
Q7

What is the difference between DNA polymerase 1 and 3?

DNA polymerase 3 is the main enzyme catalysing the 5’→3’ polymerisation of DNA strand during replication. It also has 3’→5’
exonuclease activity for proofreading. Whereas DNA polymerase 1 is the main enzyme for repair, removal of primers and filling
the gaps in the lagging strand. Apart from polymerisation and 3’→5’ exonuclease activity like DNA polymerase
 TRANSCRIPTION OF DNA

Transcription of DNA is a cellular process where the genetic information encoded in DNA is
converted into RNA. It initiates with RNA polymerase binding to the DNA at a specific promoter
region. Then, the enzyme unwinds the DNA and synthesizes a complementary RNA strand by
following the DNA template. This process continues until a termination signal is reached, leading to
the release of the newly formed RNA molecule, which carries the genetic code for protein synthesis.
Definition
Transcription is the process of copying genetic information from DNA to RNA. It involves RNA
polymerase creating a complementary RNA strand using a DNA template.
What is Transcription?
Transcription of DNA is a fundamental cellular process that converts the genetic information stored in
DNA into RNA. Both DNA and RNA are nucleic acid. While DNA stores genetic information, RNA
mostly helps in transfer and expression of information. DNA being chemically and structurally more
stable is a better genetic material. The process of transcription is governed by the principle of
complementarity. In transcription only a segment of DNA and only one of the strands is copied into
RNA.
The primary purpose of transcription is to generate RNA from the DNA sequence, with the resulting
RNA transcript containing the instructions for protein synthesis.

RNA Polymerase
RNA polymerase is an important enzyme that take part in gene expression and the transcription of
genetic information. It plays a central role in the process of transcription, where it copies DNA
sequences into RNA molecules. RNA polymerase identifies specific promoter regions on the
DNA, marking the
 initiation point for transcription. As it moves along the DNA template, RNA polymerase unwinds the
double helix and synthesizes a complementary RNA strand. This synthesized RNA, primarily
messenger RNA (mRNA), contains the instructions for protein synthesis. Multiple types of RNA
polymerases exist, with RNA polymerase II predominantly transcribing protein-coding genes in
eukaryotes. RNA polymerase regulates gene expression, enables the development and maintenance of
living organisms and the functioning and regulation of various cellular processes.

Stages of Transcription
Transcription is the process of copying genetic information from DNA into RNA.: This process
occurs in three main stages:
1. initiation
2. elongation and
3. termination

1. Initiation: This stage marks the beginning of transcription. RNA polymerase, along with
other transcription factors, recognizes and binds to the promoter region on the DNA. The
DNA double helix unwinds, exposing the template strand for transcription.
 2. Elongation: Once the RNA polymerase is bound and the DNA is unwound, the enzyme
moves along the DNA template strand. As it progresses, it synthesizes a complementary
RNA strand by incorporating ribonucleotides according to the DNA template. This stage
continues until a termination signal is encountered.

3. Termination: Transcription concludes when the RNA polymerase reaches a specific

termination signal on the DNA. This signal instructs the polymerase to release the newly
formed RNA molecule. The DNA double helix reforms, and the RNA product is freed.
The enzyme RNA polymerase latches onto the promoter sequence of a gene and starts synthesizing the
RNA strand. It keeps on adding nucleotides until it encounters the terminator sequence. Transcription
'termination' refers to the end or completion of RNA transcription process or RNA structure. This
term is often used to describe the conclusion of process of RNA synthesis.
Termination Process
Until it receives instructions to halt, RNA polymerase will continue to transcribe. Termination, which
occurs when the polymerase transcribes a DNA sequence known as a terminator, is the process of
stopping transcription. There are two primary methods of transcription termination:
a. Rho-Dependent Termination: In this mechanism, a protein called Rho factor binds to the
newly synthesized RNA and moves along it, eventually catching up to RNA polymerase. The
interaction between Rho and RNA polymerase leads to transcription termination.

b. Rho-Independent (Intrinsic) Termination: In this method, a termination signal on the

DNA template strand leads to the formation of a hairpin loop in the newly synthesized
RNA. This loop disrupts the
RNA-DNA hybrid, causing RNA polymerase to pause and then release the RNA molecule.
Contrasting Rho-Dependent and Rho-Independent Termination

Rho-Dependent
Termination Rho-Independent Termination
Intrinsic termination, also known as Rho-independent termination, is
Rho binds to ribosome- free used by prokaryotes to signal transcription termination and release the
mRNA in Rho- dependent newly synthesized RNA molecule.
termination
Hairpin loop structure does
not form Hairpin loop structure forms
Rho factor uses ATP Rho factor does not use ATP
Uracil rich region in the
transcript is absent Uracil rich region in the transcript is present

The main difference between Rho-dependent and Rho-Independent termination is the mechanism of
transcription termination. In Rho-dependent termination, the Rho protein is responsible for
termination, while the formation of a hairpin loop structure triggers Rho-independent termination.

Post transcription modifications or RNA Processing

RNA processing is a step in gene expression, particularly in eukaryotes, where the initial RNA
transcript (pre-mRNA) undergoes several modifications to become mature mRNA. These
modifications include:
1. Capping: To the 5′ end of the pre-mRNA a modified guanosine cap is added to the 5′
end of the pre-mRNA. This cap helps protect the mRNA and it is essential for
ribosome binding during translation.
2. Splicing: From the pre-mRNA, Introns (non-coding regions) are removed, and exons
(coding regions) are joined together. This process creates the mature mRNA sequence
that carries the genetic information for protein synthesis.
3. Polyadenylation: To the 3′ end of the mRNA a poly-A tail, a series of adenine
nucleotides, is added. This tail aids in mRNA stability and transport out of the nucleus.
The end product of RNA processing is mature mRNA. It is transported out of the nucleus and serves
as a template for protein synthesis at the ribosomes during translation. These processing steps ensure
that the genetic information is accurately transcribed and prepared for efficient protein production.
(This is an alternative diagram. You can draw any one.)
 Note: Additional Study

Inhibitor of Transcription
Inhibitors of transcription are molecules or compounds that can interfere with the normal process of
transcription. They reduce or prevents the synthesis of RNA from DNA. These inhibitors can have
various mechanisms of action, such as blocking the activity of RNA polymerase or disrupting the
binding of transcription factors to DNA. Some examples of transcription inhibitors includes:
1. Streptolysin: By attaching to the polymerase, it prevents the extension of nucleic acid
chains, which halts the activity of RNA polymerase within the cell.
2. Rifampicin (rifamycin): It is a medicine that fights tuberculosis. It prevents
mitochondrial RNA polymerase from working by attaching to the beta subunit of
bacterial RNA polymerase.
3. Alpha amanitin: It is an isolated eukaryotic inhibitor from Amanita phalloides that
prevents the start and elongation of RNA II polymerase.
4. Cordycepin: It demonstrates the absence of the hydroxyl moiety at the 3′ position,
which prevents RNA synthesis and transcription elongation.
5. Actinomycin D: Antibiotic has antibacterial and anticancer properties. It prevents
rRNA transcription.

FAQs on Transcription
2. What is the process of transcription?
Transcription is the process of copying genetic information from DNA to RNA. It involves RNA
polymerase binding to a DNA promoter. It synthesizes a complementary RNA strand using the DNA
template, and terminating when it reaches a specific signal.
3. Where the transcription start and terminate?
Transcription starts at the promoter region on the DNA, that is it starts at the 5′-end of the DNA
sequence. It marks the beginning of the gene to be transcribed. Transcription terminates when RNA
polymerase encounters a specific termination signal on the DNA.
4. What is the End Product of Transcription?
RNA molecule, mainly messenger RNA (mRNA), is the end product of transcription, which carries the
genetic code from DNA and serves as a template for protein synthesis during translation.
5. What are the Promoter eSquences?
Promoter sequences are specific DNA regions where RNA polymerase and transcription factors bind
to initiate transcription. These are located upstream at the 5′ end of the DNA sequence.
6. Are Enhancers Necessary for Transcription?
Enhancers are not necessary for transcription initiation. They can greatly enhance gene expression
by increasing transcription rates. They are regulatory DNA sequences that facilitate precise control
of transcription.
 RNA POLYMERASES (RNA POL/ RNA P) AND SIGMA FACTOR

What is RNA Polymerase?

RNA Polymerase, abbreviated as RNA Pol or RNAP, is an enzyme in molecular biology that
synthesises RNA from a DNA template. During the process of transcription, RNA polymerase copies
the sequence of DNA into an RNA sequence with the help of the enzyme helicase that opens up the
wounded DNA strands.

The RNA Pol not only transcribes DNA but also facilitates the process of attachment and elongation
of nucleotides, proofreads the transcribed RNA and takes part in the recognition of terminator regions.

The RNA produced by RNAP are functional mRNAs that encode protein (translation) or produce
non-coding functional RNAs, such as tRNA, rRNA and miRNA. RNA Polymerase is an important
enzyme that is found in prokaryotes, eukaryotes, as well as viruses. The size and number of subunits
in the RNAP complex are variable depending upon the type of organism.

While bacteria and archaea have a single RNA Pol, eukaryotes have five types of enzyme, each with
their designated functions.

Structure of RNA Polymerase

Roger D. Kornberg, an American biochemist, demonstrated a detailed molecular image of the RNAP
enzyme during various transcriptional stages. He was awarded a Nobel Prize for the demonstration.

In prokaryotes, the single type of RNA Polymerase enzyme is composed of a core of five subunits:
two alpha subunits (36 kDa), one beta subunit (150 kDa), one beta prime subunit (155 kDa) and a
small-sized omega subunit. The core enzyme binds with a sigma factor to form a holoenzyme. The
core enzyme forms a crab claw along the length of the DNA to be transcribed. The RNA Pol
enzymes contain metal cofactors, such as zinc and magnesium, that help in the process of
transcription.

The eukaryotic RNAPs have a similar core structure to the enzyme but also have some additional
subunits.
Components of RNA Polymerase

Bacteria

In bacteria, the RNAP enzymes produce both mRNA and non-coding RNA.

Figure 8.2.38.2.3. Prokaryotic RNA Polymerases consist of two α subunits, a β, β’, ω, and σ
subunits.

In E. coli, as with other prokaryotes, there is only one true RNA polymerase (also primase, which
makes short RNA primers for DNA replication).

 The polymerase is a multi-subunit holoenzyme comprised primarily of two α subunits, a β

subunit, a β’ subunit, an ω subunit, and a σ subunit.
 The α subunits are primarily structural, assembling the holoenzyme and
associated regulatory factors.
 The β subunit contains the polymerase activity that catalyzes the synthesis of RNA, while
the β’ subunit is used to nonspecifically bind to DNA.
 The ω subunit is involved in assembly of the holoenzyme and may also play a
role in maintaining the structural integrity of the RNA polymerase.
 Finally, there is the σ subunit, which does not stay closely associated with the core enzyme
(αββ’ω) except when helping to initiate transcription, and is used to recognize the promoter
by simultaneously decreasing the affinity of RNAP to DNA in general, but increasing the
affinity of RNAP for specific DNA promoter sequences.

Why decrease the affinity for non-specific DNA?

When the RNAP is not in use, it does not just float about in the nucleoplasm: it is bound quite tightly
along the DNA. When the sigma is bound, the decreased affinity allows the RNAP holoenzyme to
move along the DNA and scan for promoter sequences. There are multiple isoforms of the σsubunit
(such as the sigma-70 mentioned above), each of which recognizes different promoter sequences. All
isoforms perform the same basic function of properly locating the RNAP to the start of a gene, and all
isoforms only stay attached to the holoenzyme for that one transient purpose, after which they are
released (usually after transcribing about ten nucleotides).

Once the holoenzyme has recognized and bound tightly to the DNA at the promoter site, the next
step is to “melt” the DNA (breaking the H-bonds and separating the strands of the double helix) in
that area so that the RNAP can proceed downstream, read the template DNA strand, and produce the
new RNA. Often many RNA transcripts of a gene are needed to produce a large number of active
proteins in a short span of time. Highly transcriptionally active genes therefore often have multiple
RNA polymerases reading them, one right after another. Generally, an RNA polymerase only needs
to process about 15 nucleotides before there is room for another RNAP can bind the promoter and
start another transcript.

Elongation phase of transcription

Figure 8.2.48.2.4. A new RNA polymerase can begin transcribing a gene before the previous one has
finished.

The elongation phase of transcription proceeds in a 5’ to 3’ direction, which is to say that new
nucleotides are added to the 3’-OH of the growing strand. Elongation is a stochastic process in which
one of the plentiful free- floating ribonucleotides drops into the active site of RNAP opposite the
DNA template. If it is the correct nucleotide (complementary to the template), then H-bonds will
temporarily form, stabilizing the new nucleotide in place long enough for the RNAP to catalyze the
formation of a phosphodiester bond between the 3’-OH of the RNA-in-progress and the 5’- phosphate
of the nucleotide. However, if it is the incorrect nucleotide, the proper H- bonds do not form, and the
nucleotide usually dissociates from the active site before the RNAP has a chance to bind it to the
growing RNA strand. Obviously, this is not a perfect system, and in fact, the error rate for
transcription is quite high at approximately 1 in 10000 nucleotides. Fortunately, the cell generally
churns out
many copies of RNA from any given gene very quickly (approximately 80 nucleotides per second),
most of which are either error-free or have errors that do not affect the function of the end-product
protein. Furthermore, unlike DNA, in which errors of replication get carried along from one
generation of cells to the next, RNA is not a storage medium, and its transient nature means that even
mutations that severely impact the protein function only affect the few proteins translated from that
one RNA, not the proteins generated from other RNAs made from the same template gene, much less
subsequent generations.

Function of RNA Polymerase

RNAP is an important enzyme that initiates transcription in both prokaryotes and eukaryotes. It
induces specific DNA sequences on the DNA strand known as a promoter to initiate transcription.

RNA Pol can produce complementary RNA chains to the template DNA strand and can add up to 2.4
million nucleotides in eukaryotes in a process called elongation.

RNA polymerase can produce the following RNA products:

 mRNA (messenger RNA) that translates into proteins.

 RNA genes or non-coding RNA that produce RNA strands, which do not encode
proteins but take part in other activities. They include:
 tRNA (transfer RNA) that adds amino acids to the growing polypeptide chain in the
process of translation.
 rRNA (ribosomal RNA) is a component of the ribosomes that takes part in translation.
 miRNA (microRNA) regulates gene activity.
 Ribozymes are enzymatically active RNA molecules.
Holoenzyme
Holoenzyme is a complete, functional enzyme, which is catalytically active. Holoenzyme consists of
an apoenzyme together with its cofactors.
Holoenzyme contains all the subunits required for the functioning of an enzyme, e.g. DNA
polymerase III, RNA polymerase.

Holoenzyme = Apoenzyme + Cofactor

Holoenzyme is also known as a conjugate enzyme. The apoenzyme is the protein part of the
enzyme, which is enzymatically inactive without cofactors.
A cofactor is the non-protein part of the holoenzyme, which is essential for its activity. Cofactors can
be metal ions (Mg2+, Fe3+, Zn2+) or organic molecules called coenzymes.

So the catalytically active apoenzyme-cofactor complex is known as the holoenzyme or

conjugate enzyme.

Additional study (not necessary for exam)

Although RNA polymerase was discovered in 1960, the E. coli RNAP has not yet been successfully
mapped by x-ray crystallography. However, it is very similar to the RNAP of the archaean species,
Thermophilus aquaticus, which is highly stable (= easier to crystallize) and for which an x-ray
crystallographic structure has been elucidated. The data from the Taq RNAP structure and electron
microscopic analyses of E. coli RNAP produce a lobster-claw- shaped holoenzyme. The inner surface
of the claw is lined with positively charged amino acids that can interact with the negatively charged
DNA, and when the holoenzyme binds a sigma subunit, the two halves of the claw (formed mostly by
the beta and beta’ subunits) move closer together to interact with the DNA.

Rifamycins are a class of antibiotics that include rifamycin B, made by the

bacteria Streptomyces mediterranei (incidentally just one of many antibiotics derived from the
Streptomyces genus), and rifampicin, its synthetic cousin. They work by binding within the DNA-
RNA channel near the active site of RNA polymerase, which sterically prevents the addition of
nucleotides to the RNA strand. If the organism cannot transcribe RNA, it cannot use the RNA to
make the enzymes and other proteins necessary for life either, and dies. The rifamycin binding site is
highly conserved in most prokaryotes but not in eukaryotes, so the antibiotic kills bacteria
specifically with little chance of harm to eukaryotes.

Archaea

Archaea has one single type of RNAP that synthesises all RNAs. Structurally, it is very similar to
bacterial and eukaryotic RNAP, specifically RNA Pol II.
Eukaryotic RNA Polymerases and Transcription Factors

In eukaryotes, there are many types of RNA Pol enzymes that have different functions:

 RNA Pol I: It synthesises pre-rRNA subunits that form the ribosomes.

 RNA Pol II: It synthesises precursors of microRNA, snRNA and mRNA.
 RNA Pol III: It synthesises tRNA, other precursors of rRNA and some small RNAs.
 RNA Pol IV: It is found in plants and synthesises siRNA.
 RNA Pol V: It is also involved in the synthesis of siRNa in plants.

Eukaryotes have 3 different RNA polymerases in their nuclei.

1. a. Each nuclear RNA polymerase is a large protein with about 8 to 14 subunits.

MW is approximately 500,000 for each.
2. b. Each polymerase has a different function:

RNA polymerase localization synthesizes

RNA polymerase I nucleolus pre-rRNA

pre-mRNA some
RNA polymerase II nucleoplasm
snRNAs

pre-tRNA, other small RNAs some

RNA polymerase III nucleoplasm
snRNAs
The broad function and location of all the RNA polymerases is the same: read a DNA template and
transcribe an RNA copy of it; and since the DNA is found only in the nucleus, so are the
polymerases. However, the polymerases differ in exactly what kinds of RNA they produce.

 RNA Polymerase I is specialized for producing pre-rRNA (rRNA = ribosomal RNA). The
pre-rRNA is cleaved post-transcriptionally and incorporated into the ribosomes. Since
ribosomes are assembled in the the nucleolus, that is the part of the nucleus in which most
RNA Polymerase I is concentrated.
 RNA Polymerase III also makes an RNA (5S) that is incorporated into the ribosome. It also
makes other untranslated RNAs such as tRNAs and a variety of small nuclear RNAs.
 The only RNA polymerase that makes the translatable RNA (mRNA, or messenger RNA)
that most people think of when RNA is referred to generically, is RNA polymerase II. This is
the RNA polymerase that produces pre-mRNA, which after some processing, becomes
mRNA, is transported out of the nucleus, and finally translated into proteins. All of the
eukaryotic RNA polymerases are composed of two large subunits, roughly analogous to the β
and β’ subunits of prokaryotic RNAP, but instead of just three or four other subunits, there are
over a dozen smaller subunits to the eukaryotic RNA polymerase holoenzymes.
RNA polymerases in Transcription Process Initiation
Initiation of transcription is also much more complicated. Not only is there great variety in promoters
recognized by RNAP II, both RNAP I and RNAP III recognize promoters with particular structural
characteristics.
One of the most common eukaryotic RNAP II promoters is the TATA box, named for the highly
conserved motif that defines it. Although it appears similar to the Pribnow box in prokaryotes, it is
generally located further upstream from the start site, and its position is far more variable. Whereas
the Pribnow box is located at -10, the TATA box may be located closer to -30
+/- 4. Also, rather than just a sigma factor to recognize the promoter in conjunction with the
polymerase core enzyme, the eukaryotic promoter is recognized by a multi-subunit complex called
transcription factor IID (TFIID). TFIID is comprised of TATA-binding protein (TBP) and several
TBP-associated factors (TAFs).
Figure 8.3.5. Eukaryotic Transcription. An initiation complex of several transcription factors is
needed to dock the RNA Polymerase II in position to begin transcription.
This binding of the promoter by TFIID occurs independently of RNA Polymerase II, and in fact,
RNAP II will not attach to TFIID at this time. After TFIID has bound the TATA box, two more
transcription factors, TFIIA and TFIIB, attach to the TFIID as well as the nearby DNA, stabilizing
the complex. TFIIF attaches to TFIID and TFIIB to allow docking of the RNA Polymerase II. The
complex is still not ready to begin transcription: two more factors are required. TFIIE binds TFIIF
and RNAP II, and finally, TFIIH attaches to RNAP II, providing a helicase activity needed to pry
apart the two strands of DNA and allow the polymerase to read one of them. TFIIH also has another
important enzymatic activity: it is also a serine kinase that phosphorylates the carboxyl-terminal
domain (CTD) of RNA polymerase II. There are several serines in the CTD, and as they are
sequentially phosphorylated, the CTD extends like a (negatively charged) tail and helps to promote
separation between the RNAP II and the TFIID/promoter.
Elongation
Elongation of the RNA strand in eukaryotes is very similar to that in prokaryotes with the obvious
difference that transcription occurs in the nucleus rather than in the cytoplasm. Thus, in
prokaryotes, the RNA can be
 used for translation of proteins even as it is still being transcribed from the DNA! In eukaryotes, the
situation is significantly more complex: there are a number of post-transcriptional events (5’ end-
capping, 3’ polyadenylation, and often RNA splicing) that must occur before the RNA is ready to be
transported out of the nucleus and made available for translation in the cytoplasm.
Termination
Termination of eukaryotic transcription is not well-described at this writing. RNAP I appears to
require a DNA-binding termination factor, which is not analogous to the prokaryotic Rho factor,
which is an RNA binding protein. RNAP III terminates transcription without any external factor, and
this termination usually occurs after adding a series of uridine residues. However, it does not appear
to use the hairpin loop structure found in rho-independent bacterial transcription. The termination of
protein-coding RNAP II transcripts is linked to an enzyme complex that also cleaves part of the 3’
end of the RNA off, and adds a poly-A tail. However, it is not clear how the polyadenylation complex
is involved in determining the point of transcription termination, which can be over 1000 nucleotides
beyond the poly-A site (e.g. the β-globin gene in Mus musculus). Upon termination and release from
the RNAP II and template DNA, the RNA is known as the primary transcript, but must undergo post-
transcriptional processing before it is a mature messenger RNA (mRNA) ready to be exported to the
cytoplasm and used to direct translation.Eukaryotes require several other proteins, called
transcription factors, to first bind to the promoter region and then help recruit the appropriate
polymerase.

general transcription factors for RNA polymerase II.

Factors for RNA polymerase II (human cells)

Molecular
No. of Functions to
Factor Functions
mass
subunits Recruit:
(kDa)

TFIID: TBP 1 38 Recognize core promoter (TATA) TFIIB

Recognize core promoter (non- TATA);

TFIID: TAFs 12 15-250 Positive and negative regulation RNA Pol II?
 Molecular
No. of Functions to
Factor Functions
mass
subunits Recruit:
(kDa)

12, 19, Stabilize TBP-DNA binding; Anti-

TFIIA 2
35 repression

TFIIB 1 35 Select start site for RNA Pol II RNA PolII-TFIIF

RNA Pol II 12 10-220 Catalyze RNA synthesis TFIIE

Target RNA PolII to promoter;

TFIIF 2 30, 74 destabilize non-specific interactions
between PolII and DNA

Modulate TFIIH helicase, ATPase and

TFIIE 2 34, 57 kinase activities; Directly enhance TFIIH
promoter melting?

TFIIH 9 35-89 Helicase to melt promoter; CTD

kinase; promoter clearance?

Roeder, R.G. (1996) TIBS 21: 327-335.

Additional study
The eukaryotic RNA polymerases were named I, II, and III based on their elution order from ion-
exchange chromatography purification. They are also partially distinguishable by their sensitivity to
α-amanitin and related amatoxin-family mushroom poisons. RNAP I (and prokaryotic RNAP) is
insensitive to these toxins, RNAP III is somewhat sensitive (Kd ~10-6 M), and RNAP II is highly
sensitive (Kd ~10-8 M). These toxins act by binding to a site in the RNA-DNA cleft and interfering
with translocation of the RNA. That is,
there is no problem with importing a nucleotide or with attaching it to the new RNA, but the RNA
strand cannot move through the active site and allow the next nucleotide to be added.
RNA Processing

Figure 24.3.1.224.3.1.2: Eukaryotic mRNA contains introns that must be spliced out. A 5' cap and 3'
poly-A tail are also added.
 TRANSLATION: THE MECHANISM OF PROTEIN SYNTHESIS

Translation is similar in prokaryotes and eukaryotes.

1. Initiation

The initiation of protein synthesis begins with the formation of an initiation complex.

In E. coli, this complex involves the small 30S ribosome, the mRNA template, three initiation factors
that help the ribosome assemble correctly, guanosine triphosphate (GTP) that acts as an energy
source, and a special initiator tRNA carrying N-formyl-methionine(fMet-tRNAfMet) (Fig.4).

The initiator tRNA interacts with the start codon AUG of the mRNA and carries a formylated
methionine (fMet). Because of its involvement in initiation, fMet is inserted at the beginning (N
terminus) of every polypeptide chain synthesized by E. coli.

In E. coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine- Dalgarno
sequence (also known as the ribosomal binding site AGGAGG), interacts through complementary
base pairing with the rRNA molecules that compose the ribosome.
This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. At
this point, the 50S ribosomal subunit then binds to the initiation complex, forming an intact ribosome.

In eukaryotes, initiation complex formation is similar, with the following differences:

 The initiator tRNA is a different specialized tRNA carrying methionine, called Met-
tRNAi
 The eukaryotic initiation complex recognizes the 5’ cap of the eukaryotic mRNA, then tracks
along the mRNA in the 5’ to 3’ direction until the AUG start codon is recognized. At this
point, the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit.

Figure 4: Translation in bacteria begins with the formation of the initiation complex, which includes
the small ribosomal subunit, the mRNA, the initiator tRNA carrying N-formyl- methionine, and
initiation factors. Then the 50S subunit binds, forming an intact ribosome.

2. Elongation

In prokaryotes and eukaryotes, the basics of elongation of translation are the same.

In E. coli, the binding of the 50S ribosomal subunit to produce the intact ribosome forms three
functionally important ribosomal sites:
 a. The A (aminoacyl) site binds incoming charged aminoacyl tRNAs.
b. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide
bonds with the growing polypeptide chain but have not yet dissociated from their
corresponding tRNA.
c. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino
acids.

There is one notable exception to this assembly line of tRNAs: During initiation complex formation,
bacterial fMet−tRNAfMet or eukaryotic Met-tRNAi enters the P site directly without first entering
the A site, providing a free A site ready to accept the tRNA corresponding to the first codon after the
AUG.

Elongation proceeds with single-codon movements of the ribosome each called a translocation event.

During each translocation event, the charged tRNAs enter at the A site, then shift to the P site, and
then finally to the E site for removal. Ribosomal movements, or steps, are induced by conformational
changes that advance the ribosome by three bases in the 3’ direction.

Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the
carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is
catalyzed by peptidyl transferase, an RNA-based ribozyme that is integrated into the 50S ribosomal
subunit. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain.

As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the
amino acid, and is expelled.

Several of the steps during elongation, including binding of a charged aminoacyl tRNA to the A site
and translocation, require energy derived from GTP hydrolysis, which is catalyzed by specific
elongation factors.

Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning
that a 200 amino-acid protein can be translated in just 10 seconds.

3. Termination

The termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered
for which there is no complementary tRNA.

On aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes
and eukaryotes that result in the P-site amino acid detaching from its tRNA, releasing the newly made
polypeptide. The small and large ribosomal subunits dissociate from the mRNA and from each other;
they are recruited almost immediately into another translation init iation complex.

In summary, there are several key features that distinguish prokaryotic gene expression from that seen
in eukaryotes. These are illustrated in Fig.5 and listed in Fig.6.
 Figure 5: (a) In prokaryotes, the processes of transcription and translation occur simultaneously in
the cytoplasm, allowing for a rapid cellular response to an environmental cue. (b) In eukaryotes,
transcription is localized to the nucleus and translation is localized to the cytoplasm, separating these
processes and necessitating RNA processing for stability.

Protein Targeting, Folding, and Modification

During and after translation, polypeptides may need to be modified before they are biologically
active. Post-translational modifications include:

1. removal of translated signal sequences—short tails of amino acids that aid in directing a
protein to a specific cellular compartment
2. proper “folding” of the polypeptide and association of multiple polypeptide subunits, often
facilitated by chaperone proteins, into a distinct three-dimensional structure
3. proteolytic processing of an inactive polypeptide to release an active protein
component, and
4. various chemical modifications (e.g., phosphorylation, methylation, or glycosylation) of
individual amino acids.

References:
https://bio.libretexts.org/Courses/Portland_Community_College/Cascade_Microbiology/09%
3, it also has 5’→3’ exonuclease activity.