Dr.

Umayal Ramanathan College for Women, Karaikudi

Department of Biotechnology
Study material for Core – V – Cell & Molecular Biology (7BBT3C1)
Class: II B.Sc Biotechnology
Semester: III
Faculty: Dr. J. Chitra

Syllabus
Unit - IV
DNA Replication: Central dogma of molecular Biology. Mechanism of DNA replication
in Prokaryotes and Eukaryotes. Enzymes & proteins involved in DNA replication. Models of
replication. (Semi-conservative, Unidirectional, bidirectional, rolling circle mechanism).

Unit - V
Transcription – Prokaryotic & Eukaryotic Transcription. Translation: Factors involved in
translation – Mechanism of translation in Prokaryotes and Eukaryotes – Initiation – elongation –
termination.

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 Unit IV
What is DNA?
Deoxyribonucleic acid (DNA) is a nucleic acid that is made up of three components: a
deoxyribose sugar, a phosphate, and a nitrogenous base. Deoxyribonucleic acid, DNA is the
genetic material via which a cell is defined. It is a long molecule containing unique codes that
give instructions for the synthesis of all body proteins.

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DNA structure
 The structural model of DNA was initially proposed by James Watson and Francis Click.
 They found that DNA is a double-helical structure with two paired DNA strands with
complementary nucleotide sequences.
 The double-stranded DNA molecule has two spiral nucleic acid chains that are twisted into a
double helix shape. The twisting gives the DNA its compactness.
 DNA is made up of millions of nucleotides. Nucleotides are molecules that are composed of
deoxyribose sugar, with a phosphate group and a nucleobase that is attached to it.
 Each nucleotide is tightly base paired with a complementary nucleotide on the opposite strand,
i.e Adenine (A) paired with Thymine (T) or Guanine (G) paired with cytosine (C), and

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 therefore one strand’s sequence acts as a template for the new strand to be formed during
replication.
 Nucleotides are bound to each other in strands via phosphodiester bonds forming a sugar-
phosphate backbone.
 They form a bond that is between the third carbon atom on the deoxyribose sugar made up of
one sugar thus it is designated as the 3′ (three prime) and the fifth carbon atom of another sugar
on the next nucleotide as the 5′ (five prime).
 Any part of the sequence can be used to create or recognize its adjacent nucleotide sequence
during replication.
 DNA fits within the nucleus by being closely packed into tight coils known as chromatins. The
chromatins condense to form the chromosomes during cell division.
 Before DNA replication, the chromatins loosen up giving the replication machinery access to
the DNA strands.

What is DNA Replication?

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  This is a complex process that takes place during cell division, (interphase, S phase) whereby
DNA makes copies (duplicates) before the cell divides through mitosis and meiosis.
 DNA replication is a semiconservative process where a parental strand (template) is used to
synthesize a new complementary daughter strand using several protein elements which include
enzymes and RNA molecules.
 DNA replication process uses DNA polymerase as the main enzyme for catalyzing the joining
of deoxyribonucleoside 5′-triphosphates (dNTPs) forming a growing chain of DNA.
 Other proteins are also involved for initiation of the process and copying of DNA, along with
proofreading capabilities to ensure the replication process takes place accurately.
 Therefore DNA replication is a process that produces identical helices of DNA from a single
strand of the DNA molecule.
 DNA replication is an essential mechanism in enhancing cell growth, repair, and reproduction
of an organism.

Figure: The mechanism of DNA replication.

The mechanism of DNA replication
Summary: DNA replication takes place in three major steps.
1.
1. Opening of the double-stranded helical structure of DNA and separation of the strands

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2. Priming of the template strands
3. Assembly of the newly formed DNA segments.
 During the separation of DNA, the two strands uncoil at a specific site known as the origin.
With the involvement of several enzymes and proteins, they prepare (prime) the strands for
duplication.
 At the end of the process, DNA polymerase enzyme starts to organize the assembly of the new
DNA strands.
 These are the general steps of DNA replication for all cells but they may vary specifically,
depending on the organism and cell type.
 Enzymes play a major role in DNA replication because they catalyze several important stages
of the entire process.
 DNA replication is one of the most essential mechanisms of a cell’s function and therefore
intensive research has been done to understand its processes.
 The mechanism of DNA replication is well understood in Escherichia coli, which is also
similar to that in eukaryotic cells.
 In E.coli, DNA replication is initiated at the oriClocus (oriC), to which DnaA protein binds
while hydrolyzing of ATP takes place.
DNA replication enzymes and Proteins
DNA polymerase
 DNA polymerases are enzymes used for the synthesis of DNA by adding nucleotide one by
one to the growing DNA chain. The enzyme incorporates complementary amino acids to the
template strand.
 DNA polymerase is found in both prokaryotic and eukaryotic cells. They both contain several
different DNA polymerases responsible for different functions in DNA replication and DNA
repair mechanisms.
DNA Helicase enzyme
 This is the enzyme that is involved in unwinding the double-helical structure of DNA allowing
DNA replication to commence.
 It uses energy that is released during ATP hydrolysis, to break the hydrogen bond between the
DNA bases and separate the strands.

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  This forms two replication forks on each separated strand opening up in opposite
directions.
 At each replication fork, the parental DNA strand must unwind exposing new sections of
single-stranded templates.
 The helicase enzyme accurately unwinds the strands while maintaining the topography on
the DNA molecule.
DNA primase enzyme
 This is a type of RNA polymerase enzyme that is used to synthesize or generate RNA
primers, which are short RNA molecules that act as templates for the initiation of DNA
replication.
DNA ligase enzyme
 This is the enzyme that joins DNA fragments together by forming phosphodiester bonds
between nucleotides.
Exonuclease
 These are a group of enzymes that remove nucleotide bases from the end of a DNA
chain.
Topoisomerase
 This is the enzyme that solves the problem of the topological stress caused during
unwinding.
 They cut one or both strands of the DNA allowing the strand to move around each other
to release tension before it rejoins the ends.
 And therefore, the enzyme catalysts the reversible breakage it causes by joining the
broken strands.
 Topoisomerase is also known as DNA gyrase in E. coli.
Telomerase
 This is an enzyme found in eukaryotic cells that adds a specific sequence of DNA to the
telomeres of chromosomes after they divide, stabilizing the chromosomes over time.
DNA Replication Steps/Stages
Initiation
 This is the stage where DNA replication is initiated.

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  DNA synthesis is initiated within the template strand at a specific coding region site
known as origins.
 The origin sites are targeted by the initiator proteins, which recruit additional proteins
that help in the replication process to form a replication complex around the DNA origin.
 There are several origin sites on which DNA replication is initiated and they are all
known as replication forks.
 The formed replication complex contains the DNA helicase enzyme whose function is to
unwind the double helix, exposing the two strands, which act as templates for replication.
 The mechanism of DNA helicase enzyme is by hydrolyzing the ATP that is used to form
the bonds between the nucleobases, thus breaking the bond that holds the two strands.
 Additionally, during initiation DNA primase enzyme synthesizes small RNA primers that
kick-start the function of DNA polymerase.
 DNA polymerase enzyme functions by growing the new DNA daughter strand.
Elongation
 This is the phase where the DNA polymerase grows the new DNA daughter strand by
attaching to the original unzipped template strand and the initiating short RNA primer.
 The DNA polymerase is able to synthesize a new strand that matches the template, by
extending the primer via the addition of free nucleotides to the 3′ end.
 One of the templates reads in the 3′ to 5′ direction, and therefore, the DNA polymerase
synthesizes the new strand in the 5′ to 3′ direction, which is known as the leading strand.
 Along the template strand, DNA primase synthesizes a short RNA primer at the
beginning of the template in the 5′ to 3′ direction, which initiates the DNA polymerase to
continue synthesizing new nucleotides, extending the new DNA strand.
 The other template (5′ to 3′) is elongated in an antiparallel direction, by the addition of
short RNA primers which are filled with other joining fragments, forming the newly
formed lagging strand. These short fragments are known as the Okazaki fragments.
 The synthesis of the lagging strand is discontinuous since the newly formed strand is
disjointed.
 The RNA nucleotides from the short RNA primers must be removed and replaced by
DNA nucleotides, which are then joined by the DNA ligase enzyme.
Termination

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  After the synthesis and extension of both the continuous and discontinued stands, an
enzyme knows as exonuclease removes all RNA primers from the original strands.
 The primers are replaced with the right nucleotide bases.
 While removing the primers, another type of exonuclease proofread the new stands,
checking, removing, and replacing any errors formed during synthesis.
 DNA ligase enzyme joins the Okazaki fragments to form a single unified strand.
 The ends of the parent strand consist of a repetition of DNA sequences known as
telomeres which act as protective caps at the ends of chromosomes preventing the fusion
of nearby chromosomes.
 The telomeres are synthesized by a special type of DNA polymerase enzyme known as
telomerase.
 It catalyzes the telomere sequences at the end of the DNA.
 On completion, the parent and complementary strand coil into a double helical shape,
producing two DNA molecules each passing one strand from the parent molecule and one
new strand.
Okazaki fragments
 The two DNA strands run in opposite or antiparallel directions, and therefore to
continuously synthesize the two new strands at the replication fork requires that one
strand is synthesized in the 5’to3′ direction while the other is synthesized in the opposite
direction, 3’to 5′.
 However, DNA polymerase can only catalyze the polymerization of the dNTPs only in
the 5’to 3’direction.
 This means that the other opposite new strand is synthesized differently. But how?
 By the joining of discontinuous small pieces of DNA that are synthesized backward from
the direction of movements of the replication fork. These small pieces or fragments of the
new DNA strand are known as the Okasaki Fragments.
 The Okasaki fragments are then joined by the action of DNA ligase, which forms an
intact new DNA strand known as the lagging strand.
 The lagging phase is not synthesized by the primer that initiates the synthesis of the
leading strand.

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  Instead, a short fragment of RNA serves as a primer (RNA primer) for the initiation of
replication of the lagging strand.
 RNA primers are formed during the synthesis of RNA which is initiated de novo, and an
enzyme known as primase synthesizes these short fragments of RNA, which are 3-10
nucleotides long and complementary to the lagging strand template at the replication
fork.
 The Okazaki fragments are then synthesized by the extension of the RNA primers by
DNA polymerase.
 However, the newly synthesized lagging strand is that it contains an RNA-DNA joint,
defining the critical role of RNA in DNA replication.

Figure: Okazaki fragments.

Replication Fork Formation and its function
 The replication fork is the site of active DNA synthesis, where the DNA helix unwinds
and single strands of the DNA replicates.
 Several sites of origin represent the replication forks.
 The replication fork is formed during DNA strand unwinding by the helicase enzyme
which exposes the origin of replication. A short RNA primer is synthesized by primase
and elongation done by DNA polymerase.
 The replication fork moves in the direction of the new strand synthesis. The new DNA
strands are synthesized in two orientations, i.e 3′ to 5′ direction which is the leading
strand, and the 5′ to 3′ orientation which is the lagging strand.

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  The two sides of the new DNA strand (leading and lagging strand) are replicated in two
opposite directions from the replication fork.
 Therefore the replication fork is bi-directional.

Figure: DNA Replication Fork.

Leading Strand
 The leading strand is the new DNA strand that is continuously synthesized by the DNA
polymerase enzyme.
 It is the simplest strand that is synthesized during replication.
 The synthesis starts after the DNA strand has unzipped and separated. This generates a
short piece of RNA known as a primer, by the DNA primase enzyme.
 The primer binds to the 3′ end (start) of the strand, thus initiating the synthesize of the
new strand (leading strand).
 The synthesis of the leading strand is a continuous process.
The Lagging Strand

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  This is the template strand (5′ to 3′) that is synthesized in a discontinuous manner by
RNA primers.
 During the synthesis of the leading strand, it exposes small, short strands, or templates
that are then used for the synthesis of the Okasaki fragments.
 The Okasaki fragments synthesize the lagging strand by the activity of DNA polymerase
which adds the pieces of DNA (the Okasaki fragments) to the strand between the primers.
 The formation of the lagging strand is a discontinuous process because the newly formed
strand (lagging strand) is the fragmentation of short DNA strands.

Why is DNA replication important?

 DNA replication takes place during cell division and it enables the multiplication and
division of DNA by making two copies of the genome from a single parent genome.
 And therefore, its importance is in the creation of new and next copies of DNA giving
rise to two daughter cells from a single parent cell.
 Each new cell is formed with its own genome.
 This enhances heredity via reproduction and cell division.
DNA replication stress
During DNA replication, the process and the DNA genome undergoes various stress arising from
the mechanism. these stresses an result in stalled replication and stalled replication fork
formation. Several events contribute to these stresses, including;
 Unusual DNA structure
 Mismatched ribonucleotides
 Tensions arising from concurrent mechanisms of replication and transcription
 Inadequate availability of important replication factors
 Fragile sites on the replicating DNA strand
 Overexpression or constitutive activation of oncogenes
 Inaccessible chromatins
Kinase regulatory proteins such as ATM (ATM serine/threonine kinase) and ATP are proteins
that assist in alleviating replication stress. These proteins get recruited and activated by DNA
damages.

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Stalled replication forks may collapse if the regulatory proteins do not stabilize, and if and when
this happens, initiation of repairing mechanisms to reassembling of the replication fork takes
place. this helps to amend damages the damaged ends of DNA.
DNA Replication in Eukaryotes (Differences with prokaryotes)
DNA replication in prokaryotes and eukaryotes have several similar features and also
differences. This depends on the cell sizes and genome sizes.
Similarities between Prokaryotic and Eukaryotic DNA Replication
 The unwinding mechanism of DNA before replication is initiated is the same for both
Prokaryotes and eukaryotes.
 In both organisms, the DNA polymerase enzyme coordinated the synthesis of new DNA
strands.
 Additionally, both organisms use the semi-conservative replication pattern, making the
leading and lagging strands in different directions. Okasaki fragments make the lagging
strand.
 Lastly, both organisms initiate DNA replication using a short RNA primer.


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Differences between DNA replication in Eukaryotes and Prokaryotes

S.N. Eukaryotic DNA Replication Prokaryotic DNA replication

Due to the large size of eukaryotes, they Due to its small size, they have very
1.
possess 25 times more DNA minimal/little DNA

Prokaryotic cells have a single point of origin

Eukaryotic cells have multiple points of
and replication takes place in two opposite
2. origin and they use unidirectional replication
directions at the same time and it takes place
within the nucleus of the cell.
in the cell cytoplasm.

Eukaryotes have four or more types of Prokaryotic cells possess one or two types of
3.
polymerases. polymerases.

Replication of eukaryotic cells is slower Replication in prokaryotic cells is faster,

4.
taking up to 400 hours. taking up to 40 minutes.

Eukaryotes have a distinct process for Prokaryotes have circular chromosomal DNA
5. replicating the telomeres at the ends of their therefore they do not have any ends to
chromosomes. synthesize.

Eukaryotic cells only undergo DNA

6. replication during the S-phase of the cell Replication in prokaryotes takes place al
cycle.

Central Dogma of Molecular Biology- Replication, Transcription, Translation

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  DNA contains the complete genetic information that defines the structure and function of
an organism.
 Proteins are formed using the genetic code of the DNA.
 Conversion of DNA encoded information to RNA is essential to form proteins.
 Thus, within most cells, the genetic information flows from – DNA to RNA to protein.
 The flow of information is followed through three different processes which are
responsible for the inheritance of genetic information and for its conversion from one
form to another:
1. Replication: a double stranded nucleic acid is duplicated to give identical copies. This process
perpetuates the genetic information.
2. Transcription: a DNA segment that constitutes a gene is read and transcribed into a single
stranded sequence of RNA. The RNA moves from the nucleus into the cytoplasm.
3. Translation: the RNA sequence is translated into a sequence of amino acids as the protein is
formed. During translation, the ribosome reads three bases (a codon) at a time from the RNA
and translates them into one amino acid.
 This flow of information is unidirectional and irreversible.

 This explanation is the simplest way in which the Central Dogma of Molecular
Biology is interpreted.

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  In the bigger picture, the central dogma of molecular biology is an explanation of the
flow of genetic information within a biological system.
 It was first stated by Francis Crick in 1958, as
 “Once ‘information’ has passed into protein it cannot get out again. In more detail, the
transfer of information from nucleic acid to nucleic acid or from nucleic acid to protein
may be possible, but transfer from protein to protein, or from protein to nucleic acid is
impossible.”
The Dogmas
 The dogma is a framework for understanding the transfer
of sequence information between information-carrying biopolymers,
DNA and RNA (both nucleic acids), and protein.
 There are 3×3=9 conceivable direct transfers of information that can occur between these.
 The dogma classes these into 3 groups of 3:
A. Three general transfers
 It describes the normal flow of biological information: DNA can be copied to DNA
(DNA replication), DNA information can be copied into mRNA (transcription), and
proteins can be synthesized using the information in mRNA as a template (translation).
 It is believed to occur normally in most cells.
B. Three special transfers
 The special transfers describe: RNA being copied from RNA (RNA replication), DNA
being synthesised using an RNA template (reverse transcription), and proteins being
synthesised directly from a DNA template without the use of mRNA.
 Temin (1970) reported the existence of an enzyme “RNA dependent DNA polymerase”
(inverse transcriptase) which could synthesize DNA from a single stranded RNA
template.
 Baltimore (1970) also reported the activity of this enzyme in certain RNA tumour
viruses.
 This exciting finding in molecular biology gave rise to the concept of central dogma
reverse” or teminism, suggesting that the sequence of information flow is not necessarily
from DNA to RNA to protein but can also take place from RNA to DNA.

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 It is known to occur, but only under specific conditions in case of some viruses or in a
laboratory.

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 Unit V
DNA Transcription (RNA Synthesis)
DNA Transcription Definition
DNA transcription, also known as RNA synthesis is the process by which genetic
information that is contained in DNA is re-written into messenger RNA (mRNA) by an
RNA polymerase enzyme.
 The synthesized mRNA is transported out of the cell nucleus where it will later on aid in the
synthesis of proteins by the mechanism of translation.
 Regulation of mRNA production in the nucleus, the cell automatically regulated the rate of
gene expression.
 The process of transcription is aided by the RNA polymerase enzyme, which copies the right
sequences on the DNA template to produce a complementary RNA copy of the gene.
 The basic mechanism of transcription is the same in both eukaryotes and prokaryotes,
however, it may differ in a number of ways between them.
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DNA Transcription Enzymes and Function
The major enzyme used in DNA transcription is RNA polymerase. In prokaryotes, one type of
RNA polymerase enzyme is used, while in eukaryotes, three types of RNA polymerases are used
i.e RNA polymerase I, II, and III.

Figure: RNA Polymerase.

The major functions of RNA polymerase include:

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1. Formation of the initiator complex which aids in the unwinding process of the double-helical
structure of DNA
2. Synthesis and elongation of the RNA transcript by adding the nucleotide bases, Adenine (A),
Cytosine (C), Guanine (G), and Uracil (U).
3. It forms the termination sequences that stop and terminate transcription.
DNA Transcription Steps
Summary: Steps of Transcription

 50 different protein transcription factors will bind to the promoter sites, on the 5′ side of the
gene to be transcribed.
 The RNA polymerase binds to the transcription factor complex, allowing the double helix of
DNA to open up.
 The RNA polymerase then reads one strand in the 3′ to 5; direction
 In eukaryotic cells, the nucleosome in the advancing RNA polymerase (Pol II), for protein-
coding genes.
 The RNA polymerase and transcription factors complex replaces the nucleosome after DNA
has been transcribed and Pol II has moved on.
 AS the RNA polymerase moves along the DNA strand, it assembles ribonucleotides into a
strand of RNA, by utilization of triphosphate (ATP)

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  Each ribonucleotide is inserted into the growing RNA strand by a base-pairing i.e each
cytosine (C) is linked to guanine (G), while a Uracil(U) is linked to an Adenine (A).
 The RNA synthesis takes place in the 5′-3′ direction
 As each nucleoside triphosphate is added to the 3′ end of the growing strand, the two terminal
phosphates are removed.
 When transcription comes to an end, the transcript is released from the polymerase, and the
polymerase enzyme is also released from the DNA
DNA Transcription Process
 In prokaryotic cells, the entire mechanism of transcription is summarized in three stages:
Initiation, Elongation, and Termination.
 At the end of the termination in prokaryotes, the mRNA formed is ready for translation.
 Unlike in eukaryotes, after termination, an immature mRNA is formed, and therefore, more
processes are needed to form a mature mRNA which is then translated into proteins.
 Generally, the transcription process transcribes DNA into mRNA, the type of RNA that carries
the information that is needed in the synthesis of proteins.
 In eukaryotes, there are two broad steps that take place in transcription;
1.
1. Pre-messenger RNA formation using an RNA polymerase enzyme
2. Editing of pre-messenger RNA by splicing
 Pre-messenger RNA formation involves the initiation, elongation, and termination phases
which end by forming mRNA.
 The mRNA then undergoes different stages of splicing to form mature mRNA.
Formation of pre-messenger RNA

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 Figure: DNA Transcription Initiation.
 When transcription starts, DNA must unwind, aided by RNA polymerase, which catalyzes the
process.
 In transcription only one of the DNA strands is transcribed, the strand that has the initiator
sequence. this strand is known as the sense strand, while the complementary strand is known as
the antisense strand.
 mRNA that is transcribed is normally a copy of the sense strand, however, it is the antisense
strand that is transcribed.
 The ribonucleoside triphosphate (NTPs) aligns along the antisense DNA strand by base
pairing, then the RNA polymerase joins the ribonucleotides together forming a pre-messenger
RNA molecule, complementary to a region on the antisense strand.
 Transcription is completed when the RNA polymerase enzyme finds a triplet of bases read as a
stop signal. AT this stage, the DNA molecule rewinds to reform the double helix.

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 The formation of the messenger RNA (mRNA) is done in three stages: Initiation, elongation,
and termination
Initiation
Promoter and initiation in prokaryotes

Figure: Promoter and initiation in prokaryotes.

 The initiation of transcription is signaled at a region known as a promoter.
 The promoter is the site for RNA polymerase binding, such that the promoter guides the
polymerase where it should sit on the DNA in order to initiate transcription.
 RNA polymerase is the enzyme that catalyzes the mechanism of transcription.
 The RNA polymerase enzyme has a sigma (σ) factor, which is the dissociative unit, that allows
the enzyme to recognize the promoter sequence (starting point of transcription), which is
spaced between -35 and -10 regions.
 The promoter sequence is recognized by RNA polymerase’s holoenzyme subunit, by attaching
to and moving along the DNA template molecule. This forms a closed promoter complex.
 A single DNA molecule may have multiple promoter sequences or closed promoter complexes.
 The promoter which is bound with transcription factors along with the RNA polymerase forms
a complex.
 The transcription factors are regulatory proteins that control transcription rate.

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 When the RNA polymerase is bound to the promoter sequence, it denaturalizes the DNA
duplex locally, forming open promoter complex which becomes the unwound part of the
double-stranded DNA, exposing the bases on each of the two DNA strands.
Promoter and initiation in Eukaryotes

Figure: Promoter and initiation in Eukaryotes.

 In eukaryotes, the RNA polymerase does not directly attach to the promoter sequence like in
prokaryotes.
 A helper promoter known as a basal (general) transcription factor binds to the promoter first,
which helps the RNA polymerase attach to the DNA template.

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 Eukaryotes have a promoter sequence called a TATA box, which is recognized by the
transcription factors, which eventually allow the binding of the RNA polymerase.
 The TATA box has lots of As and Ts making it easy to pull the strands of DNA apart.
Elongation

Figure: DNA Transcription Elongation.

After initiating transcription, the sigma (σ) factor dissociates from the RNA polymerase.
 The template strand is read in the 3′ to 5′ direction, which means that RNA synthesis takes
place in the 5′ to 3′ direction, with the nucleoside triphosphate (NTPs) acting as substrates for
the enzyme.
 The other strand from the DNA template is known as the coding strand, because the base
sequence of the new mRNA is identical to it, except for the replacement of thiamine with
uracil base.
 The RNA polymerase catalyzes the formation of a phosphodiester bond between the adjacent
ribonucleotides.
 The energy used by the RNA polymerase is derived from splitting the high-energy triphosphate
into monophosphate, releasing the inorganic diphosphates (PPi).
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 A transcription bubble is formed and it must be maintained sine transcription takes place on the
double-stranded DNA template. The bubble moves along the DNA duplex during elongation.
 Stalling or pausing are common, which are later essential for transcription termination.
Termination
 This is the process of ending transcription, which happens when signaled by a stop sequence
known as a terminator sequence.
 This happens when the RNA polymerase transcribes the terminator sequence.
 The RNA polymerase then releases the DNA temple which unwinds back to a double-helical
structure.
Termination in bacteria
There are two termination methods in bacteria
Rho-dependent termination

Figure: Rho-dependent termination.

This is the termination process where the RNA molecule contains a binding site for a protein
known as the Rho factor, which binds to the DNA sequence. It starts to climb up the transcript
towards the RNA polymerase ad reaches the transcription bubble. At the bubble, the Rho factor
pulls the RNA transcript and the DNA template strand apart, releasing the RNA molecule and

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 terminating the transcription process. A transcription stop point sequence that is found later in
the DNA causes the RNA polymerase to stop and allow the Rho factor to catch up and terminate
the process.
Rho-independent termination

Figure: Rho-independent termination.

This process depends on a specific sequence found on the DNA template strand. During
transcription, as the RNA polymerase approaches the endpoint of the gen that is transcribed, it
reaches a region that is rich in Cytosine (C) and Guanine(G). The RNA that is transcribed from
this region folds back on itself, and the complementary C and G bind together forming a stable
hairpin that makes the RNA polymerase to stall. The hairpin is followed by a Uracil (U) in the
RNA terminator which complementary to the DNA template Adenine (A). The U-A region
forms a weak interaction with the DNA template and with the stalled RNA polymerase causes an
instability allowing the enzyme to fall off and end from the new RNA transcript.
Pre-translational mRNA processing
 In eukaryotes, the mRNA that has been transcribed is known as pre-mRNA, and therefore, it
must undergo other processes for it to mature into mature mRNA.
 These are known as the pre-translational mRNA processes. They include:
5′ Capping
 This is the addition of methylated guanine cap to the 5′ end of mRNA
 The 5′ cap helps in the recognition of the mRNA molecule by the ribosomes, and to also
protect the immature mRNA from the degradation of RNases.

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 Polyadenylation
 This is the addition of a poly (A) tail to the 3′ end of mRNA. The poly (A) tail is made up of
several molecules of adenosine monophosphate, which stabilizes RNA, because of its natural
instability.
Splicing
 This is the coding of one genetic sequence for different proteins, conserving the genetic
material.
 The process involves:
 Removing of the non-coding sequences known as introns by spliceosome excision.
 Joining of the coding sequences known as exons by ligation.
 Splicing is sequence-dependent, therefore it occurs within the transcript.
 This allows many proteins to be made from a single pre-mRNA
 At the end of the splicing process, mature mRNA will have been made.
 Mature mRNA then becomes the messenger carrier which allows protein synthesis to occur.
 Mature mRNA has open Reading Frames (ORF), a region that gets translated into proteins.
Translation on the ORFs is done in three blocks of three nucleotides known as codons.
 At the end of the 5′ and 3′ ends are untranslated regions (UTRs) which are not translated
during protein synthesis.
DNA Transcription in Eukaryotes (Difference from prokaryotes)
Transcription in eukaryotes and prokaryotes have some similarities and differences.
Similarities
Some of the common similarities include;
 DNA is used as the template in both organisms
 RNA polymerase is the main enzyme that facilitates the entire mechanism in both organisms
 The RNA molecule is the end product in both organisms
 The chemical composition of the transcript is the same in both organisms

Differences
The major differences in the transcription between eukaryotes and prokaryotes include:
Promoter:
 Prokaryotes have three promoter elements i.e -10, -35 promoters, and upstream elements

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 Eukaryotes have many different promoter elements i.e TATA box, initiator elements,
downstream core promoter, CAAT box, and the CG box
RNA polymerase:
 Prokaryotes have one type of RNA polymerase that aids in the synthesis of the RNA strand.
 Eukaryotes have three types of RNA polymerases I, II, and III that aid in the RNA strand
synthesis.
Initiation:
 The initiation complex of eukaryotes is made up of various transcription factors that dissociate
on completion of the initiation process.
 While prokaryotes do not form an initiator complex
Transcription and translation concurrence
 Another major difference is that, in prokaryotes, transcription and translation occur
simultaneously while in eukaryotes, transcriptions must be complete before the translation
mechanism is initiated.
 The RNA in eukaryotes undergoes post-transcriptional modifications like capping,
polyadenylation, and splicing to form a mature mRNA that proceeds to translation. These
processes do not take place in prokaryotes
RNA genetics
 The mRNA in prokaryotes has many different genes on the single mRNA hence they are
termed as polycistronic
 Eukaryotes have a single gene on one mRNA molecule hence termed as monocistronic.
Termination
 Termination in prokaryotes is aided by either Rho-dependent or Rho-independent factors,
while in eukaryotes, transcription is terminated by the poly-adenylated (A) signal and the
downstream terminator sequence.

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 Reverse Transcription
 This is the conversion of RNA to DNA, by which RNA acts as the template in the synthesis of
a type of DNA known as the complementary DNA (cDNA).
 The central dogma defines the mechanisms that involve DNA synthesis (replication), RNA
synthesis (transcription), protein synthesis (translation), and cDNA synthesis (reverse
transcription). Therefore, DNA codes for RNA, RNA codes for proteins, and RNA can also
code for DNA in the case of reverse transcription.
 RNA-encoded viruses undergo the reverse transcription mechanism where their genomic RNA
is converted to DNA by the aid of reverse transcriptase enzyme.
 Reverse transcription is also known as retrotranscription or retrotras. It is an extremely
erroneous process that can lead to mutations that can cause drug resistance.
 The conversion of RNA to DNA is normally desirably applied in the laboratory majorly as a
diagnostic tool for most RNA viruses such as HIV, hepatitis, influenza, coronaviruses, etc
Transcription Inhibitors
Transcription inhibitors are elements that are used to inhibit the action and mechanism of the
RNA polymerase enzyme, which hinders the process of transcription. Transcription inhibitors
are used majorly to hinder bacterial mechanisms of transcription in disease-causing pathogens.
Some of the most commonly used inhibitors include
 α-amanitin – This is an inhibitor extracted from yeast, that is selective for RNA polymerase II
and RNA polymerase III.
 Rifampicin inhibits bacterial transcription by inhibiting DNA-dependent RNA polymerase by
binding to the beta-subunit.
 8-hydroxyquinoline is also an antifungal transcription inhibitor
 Others include actinomycin D, CDK9 inhibitors such as DRB, and flavopiridol, triptolide.
 An inhibitory mechanism is histone methylation that bars the action of transcription.

Translation

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 What is Protein Synthesis?
Protein Synthesis is a process of synthesizing proteins in a chain of amino acids known as
polypeptides. It is the second part of the central dogma in genetics.
 It takes place in the ribosomes found in the cytosol or those attached to the rough endoplasmic
reticulum.
 The functions of the ribosome are to read the sequence of the codons in mRNA and the tRNA
molecules that transfer or transport or bring the amino acids to the ribosomes in the correct
sequence. However, other molecules are also involved in the process of translation such as
various enzymatic factors.
 The translation process involves reading the genetic code in mRNA to make proteins.
 The entire translation process can be summarized into three phases: Initiation, elongation, and
termination.

Figure: Central Dogma.

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Protein Synthesis Machinery

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 The translation process is aided by two major factors: A translator – this is the molecule that
conducts the translation; substrate – this is where the mRNA is translated into a new protein
(translator desk). The translation process is guided by machinery composed of:
Ribosomes
 Ribosomes are made of ribosomal RNA (rRNA) and proteins, and therefore they are also
named ribozymes because the rRNA has enzymatic activity. the rRNA has the peptidyl
transferase activity that bonds the amino acids.
 The ribosomes have two subunits of rRNA and proteins, a large subunit with three active sites
(E, P, A) which are critical for the catalytic activity of ribosomes.
Transfer RNA (tRNA)
 Each tRNA has an anticodon for the amino acid codon it carries which are complementary to
each other. For example; Lysine is coded by AAG, and therefore the anticodon that will be
carried by tRNA will be UUC, therefore when the codon AAG appears, an anticodon UUC of
tRNA will bind to it temporarily.
 When tRNA is bound to mRNA, the tRNA then releases its amino acid. rRNA then helps to
form bonds between the amino acids as they are transported to the ribosomes one by one, thus
creating a polypeptide chain. The polypeptide chain keeps growing until it reaches a stop
codon.
Protein Synthesis enzymes and functions
 Peptidyl transferase is the main enzyme used in Translation. It is found in the ribosomes with
an enzymatic activity that catalyzes the formation of a covalent peptide bond between the
adjacent amino acids.
 The enzyme’s activity is to form peptide bonds between adjacent amino acids using tRNAs
during translation.
 The enzyme’s activity uses two substrates of which one has the growing peptide chain and the
other bears the amino acid that is added to the chain.
 It is located in the large subunit of the ribosomes and therefore, the primary function of
peptidyl transferase is to catalyze the addition of amino acid residues allowing the polypeptide
chain to grow.
 The peptidyl transferase enzyme is entirely made up of RNA and its mechanism is mediated by
ribosomal RNA (rRNA), which is a ribozyme, made up of ribonucleotides.

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 In prokaryotes, the 23S subunit contains the peptidyl transferase between the A-site and the O-
site of tRNA while in eukaryotes, it is found in the 28S subunit.
Overview of the Protein Synthesis
 The ribosomal translation is initiated when the ribosomes recognize the starting point of
mRNA, where it binds a molecule of tRNA that bears a single amino acid.
 In prokaryotes, the initial amino acid in N-formylmethionine. during elongation, the second
amino acid is linked to the first one.
 The ribosome then shifts its position on the mRNA and repeats the elongation cycle.
 When the elongation process reaches the stop codon, the amino acid chain folds spontaneously
to form a protein.
 The ribosomes then split into two subunits, but later rejoin before another mRNA is translated.
 Protein synthesis is facilitated by several catalytic proteins which include initiation, elongation,
termination factors, and guanosine triphosphates (GTP).
 GTP is a molecule that releases energy when converted into guanosine diphosphate (GDP).

Protein Synthesis Steps /Process in Details

Translation Initiation
 Protein synthesis initiation is triggered by the presence of several initiation factors IF1, IF2,
and IF3, including mRNA, ribosomes, tRNA.

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 The small subunit binds to the upstream on the 5′ end at the start of mRNA. The ribosome
scans the mRNA in the 5′ to 3′ direction until it encounters the start codon (AUG or GUG or
UUG). When either of these start codons is present, it is recognized by the initiator fMet-tRNA
(N-formylMet-tRNA). This initiator factor carries the methionine (Met) which binds to the P
site on the ribosome.
 This synthesizes the first amino acid polypeptide known as N-formylmethionine. The initiator
fMet-tRNA has a normal methionine anticodon therefore it inserts the N-formylmethionine.
This means that methionine is the first amino acid that is added and appears in the chain.
 Generally, there are three steps in the initiation process of translation;
1. Initiation of the binding of mRNA to the small ribosome subunit (the 30S), stimulating the
initiator factor IF3. this dissociates the ribosomal subunits into two.
2. The initiator factor IF2 then binds to the Guanine-triphosphate (GTP) and to the initiator fMet-
tRNA to the P-site of the ribosomes.
3. A ribosomal protein splits the GTP that is bound to IF2 thus helping in driving the assembly of
the two ribosomal subunits. The IF3 and IF2 are released.

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 Figure: Diagram of Steps of Translation (Protein Synthesis).
Translation Elongation
 The elongation of protein synthesis is aided by three protein factors i.e EF-Tu, EF-Ts,
and EF-G.
 The ribosomal function is known to shift one codon at a time, catalyzing the processes that
take place in its three sites.
 For every step, a charged tRNA enters the ribosomal complex and inserts the polypeptides that
become one amino acid longer, while an uncharged tRNA departs. In prokaryotes, an amino

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 acid is added at least every 0.05 seconds, which means that about 200 polypeptide amino acids
are translated in 10 seconds.
 The bond created between each amino acid is derived from the Guanosine Triphosphate
(GTP), which is similar to Adenosine Triphosphate (ATP).
 The three sites (A, P, E) all participate in the translation process, and the ribosome itself
interacts with all the RNA types involved in translation.
 Therefore, three distinct steps are involved in translation, and these are;
1. The mediation of elongation Factor-Tu (EF-Tu) in the entry of amino-acyl-tRNAs to the A
site. This entails the binding of EF-Tu to GTP, which activates the EF-Tu-GTP complex to
bind to tRNA. The GTP then hydrolyses to GDP releasing an energy-giving phosphate
molecule, thus driving the binding of aminoacyl-tRNA to the A site. At this point the EF-Tu is
released, leaving the tRNA in the A-site.
2. Elongation factor EF-Ts then mediates the releasing of EF-Tu-GDP complex from the
ribosomes and the formation of the EF-Tu-GTP.
3. During this translocation process, the polypeptide chain on the peptidyl-tRNA is transferred to
the aminoacyl-tRNA on the A-site during a reaction that is catalyzed by a peptidyl transferase.
The ribosomes then move one codon further along the mRNA in the 5′ to 3′ direction mediated
by the elongation factor EF-G. This step draws its energy from the splitting of GTP to GDP.
Uncharged tRNA is released from the P-site, transferring newly formed peptidyl-tRNA from
the A-site to the P-site.
Translation Termination
 Termination of the translation process is triggered by an encounter of any of the three stop
codons (UAA, UAG, UGA). These triplet stop codons, however, are not recognized by the
tRNA but by protein factors known as the release factors, (RF1 and RF2) found in the
ribosomes.

 The RF1 recognizes the triplet UAA and UAG while RF2 recognizes UAA and UGA. A third
factor also assists in catalyzing the termination process and it’s known as Release factor 3
(RF3).

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 When the peptidyl-tRNA from the elongation step arrives at the P site, the release factor of the
stop codon binds to the A site. These releases the polypeptide from the P site allowing the
ribosomes to dissociate into two subunits by the energy derived from GTP, leaving the mRNA.
 After many ribosomes have completed the translation process, the mRNA is degraded allowing
its nucleotides to be reused in other transcription reactions.
Prokaryotes Protein Synthesis vs. Eukaryotes Protein Synthesis

S.N Prokaryotes

The mRNA for translation is monocistronic, The mRNA for translation is polycistronic,
1.
coding for a single gene of polypeptides thus coding for several genes of polypeptides

A single type of RNA polymerase is used to

The three types of RNA polymerase are used
2. control the synthesis of the types of RNA
for the synthesis of cellular RNA.
molecules

It involves both subunits of the ribosomes i. e

3. It involves 70S ribosomes
40S and 60S subunits.

Transcription and translation take place

4. Transcription and translation can overlap
separately hence they do not overlap.

The pre mRNA or an mRNA undergoes The mRNA doesn’t undergo any modification
5.
modification before they are translated. before translation.

They do have a special initiator complex of A special initiator tRNA Met-tRNAf or Met –
6.
tRNA. tRNA is used.

The starting amino acid is N-formyl

7. The starting amino acid is methionine.
methionine

They have a single initiation and termination They have several initiation and termination
8.
site. sites.

9. The Ribosomal Binding Site is Kozak The ribosomal binding site (RBS) on mRNA is

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 sequence that is centered around the start the Shine-Dalgarno sequence that lies -10
codon nucleotides ahead of the initiation codon.

Several initiation factors are involved in

It involves three initiation factors IF-1, IF-2,
10. initiating the synthesise of the polypetide chain
and IF-3.
i.e eIF-2, (eIF-2, eIF-2al, eIF-a2, eIF-a

There are two chain elongation factors, EF-1 There are three chain elongation factors, EF-
11.
and EF-2 Tu, EF-Ts, and EP-G.

There is a single release factor eRF for There are three release factors (RF-1 or RF-2
12. recognition of three termination codons (UAA, and RF-3) for recognition of termination
UAG, and UGA). codons.

The genetic code may differ in mitochondria The genetic code is the same in every
13.
and chloroplast. prokaryotic organism.

Protein Synthesis Inhibitors

Antimicrobial agents are used as protein synthesis inhibitors which include:
1. Puromycin
 This is an antibiotic that is an analog of the terminal aminoacyl-adenosine part of aminoacyl-
tRNA. This antibiotic inhibits protein synthesis by releasing prokaryotic polypeptides chains
before they are completely synthesized. Its mechanism is achieved by joining its amino group to
the carbonyl group of the growing polypeptide chain on the A-site forming an adduct that
dissociates from the ribosome.
 Puromycin also contains an α-amino group similar to that on the aminoacyl-tRNA, which forms
a covalently bound peptide bond with the carboxyl group of the growing peptide with puromycin
residues, thus contributing to the dissociation of the ribosomes.
2. Streptomycin
 This is a trisaccharide that has an effect on the binding activity of formyl methionyl-tRNA to
ribosomes. This prevents the correct initiation of protein synthesis.

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3. Aminoglycoside antibiotics such as neomycin, kanamycin, and gentamycin which interfere
with the decoding site in the 16s rRNA of the small subunit.
4. Chloramphenicol inhibits the activity of peptidyl transferase.
5. Erythromycin blocks translocation by binding to the 50S subunit
6. Cycloheximide is used to block peptidyl transferase in eukaryotic ribosomes and it is used as a
laboratory tool for blocking protein synthesis in eukaryotic cells.
7. Diphtheria toxin has an A fragment that catalyzes the transfer of a single side chain of EF2
which blocks the translocation of the growing polypeptide chain.

Prokaryotic Translation (Protein Synthesis)

 Translation involves translating the sequence of a messenger RNA (mRNA) molecule to a
sequence of amino acids during protein synthesis.
 It is the process in which ribosomes in the cytoplasm or ER synthesize proteins after the
process of transcription of DNA to RNA.

The Ribosomes
 Ribosomes exist normally as separate subunits that are composed of protein and rRNA.
 The subunits come together to form a ribosome when they bind to an mRNA, near its 5’ end.
 On binding to an mRNA, the ribosome reads the nucleotide sequence from the 5’ to 3’
direction, synthesizing the corresponding protein from amino acids in an N-terminal (amino-
terminal) to C-terminal (carboxyl terminal) direction.

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 Ribosomes are located in the cytosol, either freely floating or associated with the endoplasmic
reticulum.
 They serve to synthesize proteins.
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Ribosomal Sites for Protein Translation
Each prokaryotic ribosome, shown schematically, has three binding sites for tRNAs.
1. The aminoacyl-tRNA binding site (or A site) is where, during elongation, the incoming
aminoacyl-tRNA binds.
2. The peptidyl-tRNA binding site (or P site) is where the tRNA linked to the growing
polypeptide chain is bound.
3. The exit site (or E site) is a binding site for tRNA following its role in translation and prior to
its release from the ribosome.
All three sites (A, P and E) are formed by the rRNA molecules in the ribosome.

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 THE PROCESS OF TRANSLATION

Protein synthesis (or translation) takes place in three stages:

1. Initiation
2. Elongation and
3. Termination.
 During initiation, the mRNA–ribosome complex is formed and the first codon (always AUG)
binds the first aminoacyltRNA (called initiator tRNA).
 During the elongation phase, the other codons are read sequentially and the polypeptide grows
by addition of amino acids to its C-terminal end.
 This process continues until a termination codon (Stop codon), which does not have a
corresponding aminoacyl-tRNA with which to base pair, is reached.
 At this point, protein synthesis ceases (termination phase) and the finished polypeptide is
released from the ribosome.


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 Synthesis of aminoacyl-tRNA
 Synthesis of aminoacyl-tRNAs is crucially important for two reasons:
1. Each amino acid must be covalently linked to a tRNA molecule in order to take part in protein
synthesis, which depends upon the ‘adaptor’ function of tRNA to ensure that the correct amino
acids are incorporated.
2. The covalent bond that is formed between the amino acid and the tRNA is a high energy bond
that enables the amino acid to react with the end of the growing polypeptide chain to form a
new peptide bond.
For this reason, the synthesis of aminoacyl-tRNA is also referred to as amino acid activation.
 Each tRNA molecule has a cloverleaf secondary structure with the anticodon accessible at the
end of the anticodon stem loop.
 During synthesis of the aminoacyl-tRNA, the amino acid is covalently bound to the A residue
of the CCA sequence at the 3’ end.
 Each tRNA molecule carries only a single amino acid.
 The attachment of an amino acid to a tRNA is catalyzed by an enzyme called aminoacyl-
tRNA synthetase.
 A separate aminoacyl-tRNA synthetase exists for every amino acid, making 20 synthetases in
total.
The synthesis reaction occurs in two steps.
1. The first step is the reaction of an amino acid and ATP to form an aminoacyl-adenylate (also
known as aminoacyl-AMP).
2. In the second step, without leaving the enzyme, the aminoacyl group of aminoacyl-AMP is
transferred to the 3’ end of the tRNA molecule to form aminoacyl-tRNA
The overall reaction is:
Amino acid + ATP + tRNA → aminoacyl-tRNA + AMP + PPi
Initiation of Protein Synthesis
 The first codon translated in all mRNAs is the start codon or initiation codon, AUG which
codes for methionine.
 Two different tRNAs are used for the two types of AUG codon; tRNA fMet is used for the
initiation codon and is called the initiator tRNA whereas tRNA m Met is used for internal AUG
codons.

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 In prokaryotes the first amino acid of a new protein is N-formylmethionine (abbreviated fMet).
Hence the aminoacyl-tRNA used in initiation is fMet-tRNAfMet.
 A short sequence rich in purines (5’-AGGAGGU-3’), called the Shine–Dalgarno sequence,
lies 5’ to the AUG initiation codon and is complementary to part of the 16S rRNA in the small
ribosomal subunit.
 Therefore this is the binding site for the 30S ribosomal subunit which then migrates in a 3’
direction along the mRNA until it encounters the AUG initiation codon.
 Initiation of protein synthesis requires proteins called initiation factors (IFs).
 In prokaryotes, three initiation factors (IF-1, IF-2 and IF-3) are essential.
 Because of the complexity of the process, the exact order of binding of IF-1, IF-2, IF-3, fMet-
tRNAf is controversial.
Steps Involved
1. Initiation begins with the binding of IF-1 and IF-3 to the small (30S) ribosomal subunit.
 Their role is to stop the 30S subunit binding to the 50S subunit in the absence of mRNA and
fMet-tRNAf Met which would result in a nonfunctional ribosome.
2. The small subunit then binds to the mRNA via the Shine–Dalgarno sequence and moves 3’
along the mRNA until it locates the AUG initiation codon.
3. The initiator tRNA charged with N-formylmethionine and in a complex with IF-2 and GTP
(fMet-tRNAfMet/IF-2/GTP) now binds.
4. IF-3 is released.
5. The complex of mRNA, fMet-tRNAf Met, IF-1, IF-2 and the 30S ribosomal subunit is called
the 30S initiation complex.
6. The large (50S) ribosomal subunit now binds, with the release of IF-1 and IF-2 and hydrolysis
of GTP, to form a 70S initiation complex.
Elongation of Protein Synthesis
 At the start of the first round of elongation, the initiation codon (AUG) is positioned in the P
site with fMet-tRNAfMet bound to it via codon–anticodon base pairing.
 The next codon in the mRNA is positioned in the A site.
 Elongation of the polypeptide chain occurs in three steps called the elongation cycle, namely
aminoacyl-tRNA binding, peptide bond formation and translocation:
Aminoacyl-tRNA binding

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 The corresponding aminoacyl-tRNA for the second codon binds to the A site via codon–
anticodon interaction.
 Binding of the aminoacyl-tRNA requires elongation factor EF-Tu and GTP which bind as an
aminoacyl-tRNA/EF-Tu/GTP complex.
 Following binding, the GTP is hydrolyzed and the EF-Tu is released, now bound to GDP.
 Before the EF-Tu molecule can catalyze the binding of another charged tRNA to the ribosome,
it must be regenerated by a process involving another elongation factor, EF-Ts.
This regeneration is called the EF-Tu–EF-Ts exchange cycle.
 First, EF-Ts binds to EF-Tu and displaces the GDP. Then GTP binds to the EF-Tu and
displaces EF-Ts. The EF-Tu-GTP is now ready to take part in another round of elongation.
Peptide bond formation
 The second step, peptide bond formation, is catalyzed by peptidyl transferase.
 In this reaction the carboxyl end of the amino acid bound to the tRNA in the P site is
uncoupled from the tRNA and becomes joined by a peptide bond to the amino group of the
amino acid linked to the tRNA in the A site.
Translocation
 In the third step, a complex of elongation factor EF-G (also called translocase) and GTP (i.e.
EF-G/GTP) binds to the ribosome.
 Three concerted movements now occur, collectively called translocation:
1. the deacylated tRNA moves from the P site to the E site
2. the dipeptidyl-tRNA in the A site moves to the P site, and
3. the ribosome moves along the mRNA (5’ to 3’) by three nucleotides to place the next codon in
the A site.
 During the translocation events, GTP is hydrolyzed to GDP and inorganic phosphate, and EF-
G is released ready to bind more GTP for another round of elongation.
 After translocation, the A site is empty and ready to receive the next aminoacyltRNA.
 The A site and the E site cannot be occupied simultaneously. Thus the deacylated tRNA is
released from the E site before the next aminoacyl-tRNA binds to the A site to start a new
round of elongation.

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 Elongation continues, adding one amino acid to the C-terminal end of the growing polypeptide
for each codon that is read, with the peptidyl-tRNA moving back and forth from the P site to
the A site as it grows.
Termination of Protein Synthesis
 Eventually, one of three termination codons (also called Stop codons) becomes positioned in
the A site. These are UAG, UAA and UGA.
 Unlike other codons, prokaryotic cells do not contain aminoacyl-tRNAs complementary to
 Stop codons. Instead, one of two release factors (RF-1 and RF-2) binds instead.
 RF-1 recognizes UAA and UAG whereas RF-2 recognizes UAA and UGA. A third release
factor, RF-3, is also needed to assist RF-1 or RF-2 interaction with the ribosome. Thus either
RF-1 + RF-3 or RF-2 + RF-3 bind depending on the exact termination codon in the A site.
 RF-1 (or RF-2) binds at or near the A site whereas RF-3/GTP binds elsewhere on the
ribosome.
 The release factors cause the peptidyl transferase activity to transfer the polypeptide to a water
molecule instead of to aminoacyl-tRNA, effectively cleaving the bond between the polypeptide
and tRNA in the P site.
The free polypeptide now leaves the ribosome, followed by the mRNA and free tRNA, and the
ribosome dissociates into 30S and 50S subunits ready to start translation again.

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 Protein Synthesis In Eukaryotes
Protein Synthesis involves translating the sequence of a messenger RNA (mRNA) molecule to a
sequence of amino acids during protein synthesis. It is the process in which ribosomes in the
cytoplasm or ER synthesize proteins after the process of transcription of DNA to RNA.
The Ribosomes

 Ribosomes exist normally as separate subunits that are composed of protein and rRNA.
 Eukaryotic ribosomes are larger (the 80S) and more complex than prokaryotic ribosomes
(70S).
 The subunits come together to form a ribosome when they bind to an mRNA, near its 5’ end.
 On binding to an mRNA, the ribosome reads the nucleotide sequence from the 5’ to 3’
direction, synthesizing the corresponding protein from amino acids in an N-terminal (amino-
terminal) to C-terminal (carboxyl-terminal) direction.
 Ribosomes are located in the cytosol, either freely floating or associated with the endoplasmic
reticulum.
 They serve to synthesize proteins.
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Geoscience Research

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 Ribosomal Sites for Protein Synthesis
Each prokaryotic ribosome, shown schematically, has three binding sites for tRNAs.
1. The aminoacyl-tRNA binding site (or A site) is where, during elongation, the incoming
aminoacyl-tRNA binds.
2. The peptidyl-tRNA binding site (or P site) is where the tRNA linked to the growing
polypeptide chain is bound.
3. The exit site (or E site) is a binding site for tRNA following its role in translation and prior to
its release from the ribosome.
All three sites (A, P, and E) are formed by the rRNA molecules in the ribosome.

Protein Synthesis Process

The overall mechanism of protein synthesis in eukaryotes is basically the same as
in prokaryotes.
However, there are some significant differences:
 Whereas a prokaryotic ribosome has a sedimentation coefficient of the 70S and subunits of
30S and 50S, a eukaryotic ribosome has a sedimentation coefficient of 80S with subunits of
40S and 60S.
 The composition of eukaryotic ribosomal subunits is also more complex than prokaryotic
subunits but the function of each subunit is essentially the same as in prokaryotes.

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 In eukaryotes, each mRNA is monocistronic that is, discounting any subsequent post-
translational cleavage reactions that may occur; the mRNA encodes a single protein. In
prokaryotes, many mRNAs are polycistronic that is they encode several proteins. Each coding
sequence in a prokaryotic mRNA has its own initiation and termination codons.
 Initiation of protein synthesis in eukaryotes requires at least nine distinct eukaryotic initiation
factors (eIFs) compared with the three initiation factors (IFs) in prokaryotes.
 In eukaryotes, the initiating amino acid is methionine, not N-formylmethionine as in
prokaryotes.
 As in prokaryotes, a special initiator tRNA is required for initiation and is distinct from the
tRNA that recognizes and binds to codons for methionine at internal positions in the mRNA.
When charged with methionine ready to begin initiation, this is known as Met-tRNAimet
 The main difference between initiation of translation in prokaryotes and eukaryotes is that in
bacteria, a Shine–Dalgarno sequence lies 5’ to the AUG initiation codon and is the binding site
for the 30S ribosomal subunit.
 In contrast, most eukaryotic mRNAs do not contain Shine–Dalgarno sequences. Instead, a 40S
ribosomal subunit attaches at the 5’ end of the mRNA and moves downstream (i.e. in a 5’ to 3’
direction) until it finds the AUG initiation codon. This process is called scanning.
 Prokaryotic translation requires no helicase, presumably because protein synthesis in bacteria
can start even as the mRNA is still being synthesized whereas, in eukaryotes, transcription in
the nucleus and translation in the cytoplasm are separate events that allow time for mRNA
secondary structure to form.
Protein synthesis (or translation) takes place in three stages:
1. Initiation
2. Elongation and
3. Termination

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 Initiation of Protein Synthesis

 The first step is the formation of a pre-initiation complex consisting of the 40S small ribosomal
subunit, Met-tRNAimet, eIF-2, and GTP.
 The pre-initiation complex binds to the 5’ end of the eukaryotic mRNA, a step that requires
eIF-4F (also called cap-binding complex) and eIF-3.
 The eIF-4F complex consists of eIF-4A, eIF-4E, and eIF-4G; eIF-4E binds to the 5’ cap on the
mRNA whilst eIF-4G interacts with the poly (A) binding protein on the poly (A) tail.
 The eIF-4A is an ATP-dependent RNA helicase that unwinds any secondary structures in the
mRNA, preparing it for translation.
 The complex then moves along the mRNA in a 5’ to 3’ direction until it locates the AUG
initiation codon (i.e. scanning).
 The 5’ untranslated regions of eukaryotic mRNAs vary in length but can be several hundred
nucleotides long and may contain secondary structures such as hairpin loops. These secondary
structures are probably removed by initiation factors of the scanning complex.

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 The initiation codon is usually recognizable because it is often (but not always) contained in a
short sequence called the Kozak consensus (5’-ACCAUGG-3’).
 Once the complex is positioned over the initiation codon, the 60S large ribosomal subunit
binds to form an 80S initiation complex, a step that requires the hydrolysis of GTP and leads to
the release of several initiation factors.
Elongation of Protein Synthesis

 Elongation depends on eukaryotic elongation factors.

 Three elongation factors, eEF-1A, eEF-IB, and eEF-2, are involved which have similar
functions to their prokaryotic counterparts EF-Tu, EF-Ts and EF-G.
 At the end of the initiation step, the mRNA is positioned so that the next codon can be
translated during the elongation stage of protein synthesis.
 The initiator tRNA occupies the P site in the ribosome, and the A site is ready to receive an
aminoacyl-tRNA.

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 During chain elongation, each additional amino acid is added to the nascent polypeptide chain
in a three-step microcycle.
 The steps in this microcycle are:
1. Positioning the correct aminoacyl-tRNA in the A site of the ribosome,
2. Forming the peptide bond and
3. Shifting the mRNA by one codon relative to the ribosome.
 Although most codons encode the same amino acids in both prokaryotes and eukaryotes, the
mRNAs synthesized within the organelles of some eukaryotes use a variant of the genetic
code.
 During elongation in bacteria, the deacylated tRNA in the P site moves to the E site prior to
leaving the ribosome. In contrast, although the situation is still not completely clear, in
eukaryotes the deacylated tRNA appears to be ejected directly from the ribosome.
Termination of Protein Synthesis

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 Termination of elongation depends on eukaryotic release factors.
 In eukaryotes, eukaryotic release factor eRF-1 recognizes all three termination codons (UAA,
UAG, and UGA) and, with the help of protein eRF-3, terminates translation.
 Upon termination, the ribosome is disassembled and the completed polypeptide is released.
Transcription Vs Translation
Transcription Definition
Transcription is the process where the genetic information on a DNA strand is transferred
into an RNA strand by a series of polymerization reactions catalyzed by enzymes called
DNA-dependent RNA polymerases.
 This is the first step of gene expression where the information is passed on from one structure
to another.

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 During transcription, RNA molecules are initiated, elongated, and terminated. The RNA
formed is the non-genetic RNA.
 Transcription occurs as a prerequisite for translation and occurs when there is a need for a
particular gene product at a specific time for a specific tissue.
 Only one strand of DNA, called the template strand, is replicated during transcription, and the
resulting RNA strands are single-stranded messenger RNA (mRNA).
 Transcription of a gene occurs near the chromosomal site of the gene, which is usually a short
segment of the chromosome.
 The overall process of transcription is a highly controlled process catalyzed and regulated by
the enzyme DNA-dependent RNA polymerase.
 The first step is the recognition of specific DNA sequences termed promoter sequences that
signify the beginning of the gene.
 It is then followed by the separation of two strands of DNA and replication of one of the
strands by the RNA polymerase.
 The RNA polymerase in eukaryotes is different and complex than that in prokaryotes.
 The sequence formed after replication is complementary to the template sequence as the
process follows the complementary base pairing rules of DNA, except the thymine is replaced
by uracil.
 In prokaryotes, the overall process is regulated by proteins that function as signals or operators
and terminate the process by physically blocking the RNA polymerase once the process is
complete.
 In eukaryotes, various proteins termed as transcription factors are involved in the regulation of
transcription.
 Besides, the post-transcriptional modification also takes place in eukaryotes where the pre-
mRNA (the result of transcription) is edited by the process of splicing before the mature
mRNA reaches ribosomes for translation.
 The mRNA thus produced acts as a blueprint for protein synthesis during the process of
translation.
 Depending on the sequence of DNA chosen for transcription, rRNA and tRNA synthesis also
occur.

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 Transcription occurs in the nucleus of eukaryotes and cytoplasm of prokaryotes where the
enzymes and transcriptional factors are available.
 It is inhibited by some antibiotics like rifampicin and 8-Hydroxyquinoline.
 The process can be detected by methods like RT-PCR, DNA microarray, in-situ hybridization,
and northern blotting.
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Translation Definition
The translation is the process of protein synthesis where the information on RNA is
expressed in the form of polypeptide chains.
 It is the second and last step of gene expression where the information encoded on the mRNA
sequence results in an amino acid sequence.
 The starting point of translation is the mRNA formed from the process of transcription of a
particular DNA sequence. Thus, translation follows transcription.
 Even though the information on the mRNA is utilized to produce the amino acid sequence,
other forms of RNA like tRNA is also required for the process.
 Like transcription, translation is also controlled by various factors and enzymes where the most
important enzyme is the aminoacetyl tRNA synthetase.
 Translation begins with the initiation step, which involves the binding of mRNA to the
ribosomes, followed by the transfer and binding of activated amino acid to the tRNA.
 The second step is elongation, where two amino acids are joined by the peptide bond as the
mRNA and ribosomes move with respect to one another to ensure the translation of codons
successively.
 Once all the codons are translated, the resultant polypeptide sequence is dissociated from the
translation complex, and the ribosomes are also released to begin another process of
translation.
 Termination is followed by post-translation modification where the polypeptide needs to be
folded to obtain the three-dimensional configuration. This occurs in the endoplasmic reticulum
and Golgi apparatus of the cell, and thus, the polypeptide chains are moved to those organelles.
 Other modifications are chemical in nature and involve the attachment of functional groups to
the peptide sequence.

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 The process of translation is regulated by the binding of ribosomal subunits to the translation
complex. The ribosomes function as enzymes for the regulation of various steps.
 In eukaryotes, translation occurs in the ribosomes associated with the endoplasmic reticulum
whereas, in prokaryotes, it occurs in the cytoplasm.
 The translation is inhibited by antibiotics like tetracycline, chloramphenicol, streptomycin,
erythromycin, anisomycin, cycloheximide, etc.
 Similarly, the process of translation can be detected by methods like western blotting,
immunoblotting, enzyme assay, Protein sequencing, etc.

Transcription vs Translation (Table Form)

Basis for
Transcription Translation
Comparison

Transcription is the process where the

genetic information on a DNA strand
is transferred into an RNA strand by a
Definition
series of polymerization reactions
catalyzed by enzymes called DNA-
dependent RNA polymerases.
The translation is the process of protein
synthesis where the information on RNA
is expressed in the form of polypeptide
chains.

Gene Transcription is the first step in gene The translation is the second and final step
expression expression. of gene expression.

Transcription occurs before

Occurs Translation occurs after transcription.
translation.

The precursor of transcription is the The precursor of translation is the mRNA

Precursor
non-coding or antisense DNA strand. produced from transcription.

The raw material of transcription is

The twenty amino acids are the raw
Raw material the four base pairs of RNA; adenine,
materials of translation.
guanine, uracil, and cytosine.

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 The recognition of specific DNA
sequences termed promoter sequences The binding of mRNA initiates the
Initiation
initiates transcription that signifies the translation to the ribosomes.
beginning of the gene.

The elongation of RNA sequences

occurs by the binding of The elongation of protein occurs by the
Elongation
complementary base pairs to the new binding of amino acids.
sequence.

The product of transcription is the The product of translation is the peptide

Product mRNA molecule which is sequences encoded from the mRNA
complementary to the DNA strand. sequence.

Transcription results in the synthesis Translation results in the synthesis of

Synthesis of
of RNA sequences. proteins.

Transcription occurs in the nucleus of

Translation occurs in the cytoplasm of
eukaryotes and in the cytoplasm of
Site prokaryotes and in the ribosomes on the
prokaryotes, where the enzymes and
endoplasmic reticulum in eukaryotes.
regulation factors are present.

The major enzymes that are The major enzyme that is responsible for
Enzymes responsible for transcription are translation is aminoacetyl tRNA
DNA-dependent RNA polymerase. synthetase.

Transcription is regulated by various Translational control is mainly brought

Regulation transcriptional factors in eukaryotes out by the binding of ribosomal units to
and by operons in prokaryotes. the translation complex.

Post-event Post-transcriptional modifications Post-translational modifications involve

modifications include the editing of pre-mRNA (the the folding of polypeptide chains to obtain
result of transcription) by the process the three-dimensional configuration.

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 of splicing before the mature mRNA
reaches ribosomes for translation

The process can be detected by Similarly, the process of translation can be

methods like RT-PCR, DNA detected by methods like western blotting,
Detection
microarray, in-situ hybridization, and immunoblotting, enzyme assay, Protein
northern blotting. sequencing, etc.

The translation is inhibited by antibiotics

It is inhibited by some antibiotics like like tetracycline, chloramphenicol,
Inhibition
rifampicin and 8-Hydroxyquinoline. streptomycin, erythromycin, anisomycin,
cycloheximide, etc.

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 Post Translational Modification
 Post translational modifications refer to any alteration in the amino acid sequence of the
protein after its synthesis.
 It may involve the modification of the amino acid side chain, terminal amino or carboxyl group
by means of covalent or enzymatic means following protein biosynthesis.
 Generally, these modifications influence the structure, stability, activity, cellular localization or
substrate specificity of the protein.
 Post translational modification provides complexity to proteome for diverse function with
limited number of genes.

 Location
Post-translational modifications (PTMs) mainly occur in the endoplasmic reticulum of the cell
but sometimes continue in the golgi bodies as well.
1

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Post-translational processing

After synthesis is completed, proteins can be modified by various methods such as

phosphorylation, glycosylation, ADP ribosylation, hydroxylation, and addition of other groups.

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1. Proteolysis
As the newly synthesized protein is released in the lumen of the ER, signal peptidases cleave
peptide sequence. Apart from signal peptide, some polypeptide sequence of the protein is also
cleaved resulting in the final sequence.
Example:
Insulin is synthesized in the cells in its inactive form which cannot perform its function. Post
translational modifications ensure proper function which involves the removal of the part of
protein to convert it into a three dimensional and fully active form.
2. Phosphorylation
Phosphoryalation is the addition of one or more phosphate groups to the protein. Post
Translational Phosphorylation is one of the most common protein modifications that occur in
animal cells. Majority of phosphorylation occurs as a mechanism to regulate the biological
activity of a protein. In animal cells Serine, tyrosine and thereonine are the amino acids that
subjected to the phosphorylation.
3. Glycosylation
Glycosylation is the addition of carbohydrate molecules to the polypeptide chain and modifying
it into glycoproteins. Many of the proteins that are destined to become a part of plasma
membrane or to be secreted from the cell, have carbohydrate chains attached to the amide
nitrogen of asparagine(N linked) or the hydroxyl groups of serine, threonine(O linked). N
glycosylation occurs in ER and O glycosylation occurs in the golgi complex.
4. Sulfation
Sulfate modidication takes place by the addition of sulphate molecules and these modifications
of proteins occurs at tyrosine residues. Tyrosine sulfation accomplished via the activity of
tyrosylproteinsulfotransferases (TPST) which are membrane associated enzymes of trans-Golgi
network. There are two known TPSTs. TPST-1 TPST-2 The universal phosphate donor is 3’-
phosphoadenosyl- 5’-phosphosulphate (PSPA).
5. Methylation
The transfer of one-carbon methyl groups to nitrogen or oxygen to amino acid side chains
increases the hydrophobicity of the protein and can neutralize a negative amino acid charge when
bound to carboxylic acids. Methylation is mediated by methyltransferases and S-adenosyl
methionine (SAM) is the primary methyl group donor.

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6. Hydroxylation
The biological process of addition of a hydroxy group to a protein amino acid is called
Hydroxylation. Protein hydroxylation is one type of PTM that involves the conversion of –CH
group into –COH group and these hydroxylated amino acids are involved in the regulation of
some important factors called transcription factors. Among the 20 amino acids, the two amino
acids regulated by this method are proline and lysine.
7. Others
a) SUMOylation
SUMO (small ubiquitin related modifier) proteins are 100 amino acid residue proteins which
bind to the target protein in the same way as ubiquitin. They also confer the transcription
regulatory activity of the protein and help in the transport of the target protein from cytosol to the
nucleus.
b) Disulfide bond formation
Stabilizes protein structure and involved in redox processes.
c) Lipidylation, Acetylation, Prenylation etc.

Significance
Proteins are synthesized by ribosomes translating mRNA into polypeptide chains, which may
then undergo modifications to form the mature protein product.
Post-translational modifications of proteins, which are not gene- template based, can regulate the
protein functions, by causing changes in protein activity, their cellular locations and dynamic
interactions with other proteins.
PTMs have significant biological functions which include:
 Aids in proper protein folding – few lectin molecules called calnexin binds to glycosylated
proteins and assist in its folding.
 Confers stability to the protein- glycosylation can modify the stability of the protein by
increasing protein half-life.
 It protects the protein against cleavage by proteolytic enzyme by blocking the cleavage sites.
 Protein sorting or translocation- If phosphorylated mannose residues are present in the protein
it always goes to lysosome.

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 It regulates protein activity and function- phosphorylation of protein is a reversible PTM which
activates the protein.
 Acetylation regulates many diverse functions, including DNA recognition, protein-protein
interaction and protein stability.
 Redox-dependent PTM of proteins is emerging as a key signaling system conserved through
evolution, influences many aspects of cellular homeostasis.
 PTMs are important components in cell signaling, as for example when prohormones are
converted to hormones.
 It significantly increases the diversity and complexity in the proteome.

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