Transcription Ribosomes are the structures where polypeptides (proteins) are

built. They are made up of protein and RNA (ribosomal RNA, or

Transcription is the first step of gene expression. During this
rRNA). Each ribosome has two subunits, a large one and a small one,
process, the DNA sequence of a gene is copied into RNA. Before
which come together around an mRNA. The ribosome provides a set
transcription can take place, the DNA double helix must unwind
of handy slots where tRNAs can find their matching codons on the
near the gene that is getting transcribed. The region of opened-up
mRNA template and deliver their amino acids. These slots are called
DNA is called a transcription bubble. Transcription uses one of the
the A, P, and E sites. Not only that, but the ribosome also acts as an
two exposed DNA strands as a template; this strand is called the
enzyme, catalyzing the chemical reaction that links amino acids
template strand. The RNA product is complementary to the
together to make a chain.
template strand and is almost identical to the other DNA strand,
called the non-template (or coding) strand. The site on the DNA Transcription in Prokaryotes
from which the first RNA nucleotide is transcribed is called the +1
site or the initiation site. Nucleotides that come before the initiation
site are given negative numbers and said to be upstream.
Nucleotides that come after the initiation site are marked with
positive numbers and said to be downstream.

Translation

Two types of molecules with key roles in translation are tRNAs and
ribosomes.

Transfer RNAs (tRNAs)

Transfer RNAs, or tRNAs, are molecular "bridges" that connect transcription is an enzymatic process. the mechanism of
mRNA codons to the amino acids they encode. One end of each transcription completes in three major steps
tRNA has a sequence of three nucleotides called an anticodon,
1. Initiation:
which can bind to specific mRNA codons. The other end of the tRNA
carries the amino acid specified by the codons.  closed complex formation
 Open complex formation
There are many different types of tRNAs. Each type reads one or a
 Tertiary complex formation
few codons and brings the right amino acid matching those codons.
2. Elongation
Ribosomes
3.Termination:
  Rho- dependent
 Rho-independent
1. Initiation
The transcription is initiated by RNA polymerase holoenzyme from a
specific point called promotor sequence. The core enzyme bind to
specific sequence on template DNA strand called promotor. The
binding of core polymerase to promotor is facilitates and specified
by sigma (σ) factor.
These subunits assemble every time a gene is transcribed, and they
disassemble once transcription is complete. Each subunit has a
unique role; the two α-subunits are necessary to assemble the
polymerase on the DNA; the β-subunit binds to the ribonucleoside
triphosphate that will become part of the nascent mRNA molecule;
and the β‘ subunit binds the DNA template strand. The fifth subunit,
σ, is involved only in transcription initiation. Without σ, the core
enzyme would transcribe from random sites
A promoter is a DNA sequence onto which the transcription
machinery binds and initiates transcription. In most cases,
promoters exist upstream of the genes they regulate. The specific
sequence of a promoter is very important because it determines
whether the corresponding gene is transcribed all the time, some of
the time, or infrequently. Although promoters vary among
prokaryotic genomes, a few elements are conserved. At the -10 and
-35 regions upstream of the initiation site, there are two promoter
consensus sequences, or regions that are similar across all
promoters and across various bacterial species. Binding of RNA
polymerase holoenzyme to the promotor sequence form closed
complex
The -10 consensus sequence, called the -10 region, is TATAAT. The
-35 sequence, TTGACA, is recognized and bound by σ. Once this
interaction is made, the subunits of the core enzyme bind to the
site. The A–T-rich -10 region facilitates unwinding of the DNA
template, and several phosphodiester bonds are made. The
transcription initiation phase ends with the production of abortive
transcripts, which are polymers of approximately 10 nucleotides
that are made and released. So that open complex is formed. This
changing from closed complex to open complex is called
isomerization.
RNA polymerase starts synthesizing nucleotide. It doesnot require
the help of primase. If the enzyme synthesize short RNA molecules
of less than 10 bp, it does not further elongates which is called
abortive initiation. When the RNA polymerase manage to synthesize
RNA more than 10 bp long, it eject the σ 3.2 region and RNA further
elongates and exit from RNA exit channel. This is the formation of
tertiary complex.

2. Elongation
The transcription elongation phase begins with the release of the
σsubunit from the polymerase. The dissociation of σ allows the core
RNA polymerase enzyme to proceed along the DNA template,
synthesizing mRNA in the 5’ to 3’ direction at a rate of
approximately 40 nucleotides per second. As elongation proceeds,
the DNA is continuously unwound ahead of the core enzyme and
rewound behind it. Since the base pairing between DNA and RNA is
not stable enough to maintain the stability of the mRNA synthesis
components, RNA polymerase acts as a stable linker between the
DNA template and the nascent RNA strands to ensure that
elongation is not interrupted prematurely.
Free NTPs are added sequentially to the 3’-OH of the nascent RNA
strand.
(NMP)n + NTP (NMP)n+1 + PP
The synthesized RNA exit from RNA exit channel. The synthesized
RNA is proof reads by Hydrolytic editing. For this the polymerase
back track by one or more nucleotide and cleave the RNA removing
the error and synthesize the correct one. The Gre factor enhance
this proof reading process. Pyrophospholytic editing another
mechanism of removing altered nucleotide.
Proof reading
Pyrophosphorolytic editing：The enzyme uses its active site ,in a
simple back-reaction, to catalyze the removal of an incorrectly
inserted ribonucleotide, by reincorporation of PPi. When removing
bases, the enzyme spends longer hovering over mismatches than
matches, so removes the former more frequently.
Hydrolytic editing: The polymerase backtracks by one or more
nucleotides and cleaves the RNA product, removing the error-
containing sequence. The Gre factors enhances the hydrolytic
editing function,serves as elongation stimulating factors as well.
3. Termination
Rho independent:
In this mechanism, transcription is terminated due to specific
sequence in terminator DNA.The terminator DNA contains invert
repeat which cause complimentary pairing as transcript RNA form
hair pin structure.This invert repeat is followed by larger number of
TTTTTTTT(~8 bp) on template DNA. The uracil appear in RNA. The
load of hair pin structure is not tolerated by A=U base pair so the
RNA get separated from transcription bubble.
 Steps in translation:

Activation of aminoacids:

The activation of amino acids take place in cytosol. The activation of

amino acids is catalyzed by their aminoacyl tRNA synthetases.

All the 20 amino acids are activated and bound to 3’ end of their
specific tRNA in the presence of ATP and Mg++.

The N-formylated methionine is chain initiating amino acid in

bacteria whereas methionine is chain initiating amino acid in
eukaryotes.

Methionine is activated by methionyl-tRNA synthetase. For N-

formylmethionine two types of tRNA are used ie. tRNAmet and
tRNAfmet.
Rho depended:
Similarly, all 20 aminoacids are activated (amino acyl-AMP enzyme
In this mechanism, transcription is terminated by rho (ρ) protein.It is complex) and then bound to their specific tRNA forming Aminoacyl
ring shaped single strand binding ATpase protein. The rho protein tRNA.
bind the single stranded RNA as it exit from polymerase enzyme
complex and hydrolyse the RNA from enzyme complex. The rho
protein does not bind to those RNA whose protein is being
translated. Rather it bind to RNA after translation. In bacteria
transcription and translation occur simultaneously so the rho
protein bind the RNA after translation has completed but
transcription is still ON.

Initiation:
Translation in prokaryotes
In the first step, initiation factor-3 (IF-3) binds to 30S ribosomal unit.
It is the process of synthesis of protein by encoding information on
mRNA. Protein synthesis requires mRNA, tRNA, aminoacids,
ribosome and enzyme aminoacyl tRNA synthase
Then mRNA binds to 30S ribosomal subunit in such a way that AUG iii. Ribosome translocation:
codon lie on the peptidyl (P) site and the second codon lies on
After peptide bond formation ribosome moves one codon ahead
aminoacyl (A) site.
along 5’-3’ direction on mRNA, so that dipeptide-tRNA appear on P-
The tRNA carrying formylated methionine ie. FMet–tRNA FMet is site and next codon appear on A-site.
palced at P-site. This specificity is induced by IF-2 with utilization of
The uncharged tRNA exit from ribosome and enter to cytosol.
GTP. The IF-1 prevent binding of FMet–tRNAF Met is in A-site.
The ribosomal translocation requires EF-G-GTP (translocase
Shinedalgrno sequence in the mRNA guide correct positioning of
enzyme) which change the 3D structure of ribosome and catalyze
AUG codon at P-site of 30S ribosome.
5’-3’ movement.
After binding of FMet–tRNAFMet on P-site, IF-3, IF-2 and IF-1 are
The codon on A-site is now recognized by other aminoacyl-tRNA as
released so that 50S ribosomal unit bind with 30S forming 70S
in previous.
sibosome. The exit site is located in 50S.
The dipeptide on P-site is transferred to A-site forming tripeptide.
3. Elongation:
This process continues giving long polypeptide chain of aminoacids.
i. Binding of AA-tRNA at A-site:
4. Termination:
The 2nd tRNA carrying next aminoacid comes into A-site and
recognizes the codon on mRNA. This binding is facilitated by EF-TU The peptide bond formation and elongation of polypeptide
and utilizes GTP. continues until stop codon appear on A-site.
After binding, GTP is hydrolysed and EF-TU-GDP is releasd If stop codon appear on A-site it is not recognized by t-RNA carrying
amino acids because stop codon donot have anticodon on mRNA.
EF=TU-GDP then and enter into EF-TS cycle.
The stop codon are recognized by next protein called release factor
ii. Peptide bond formation:
(Rf-1, RF-2 and RF-3) which hydrolyses and cause release of all
The aminoacid present in t-RNA of P-site ie Fmet is transferred to t- component ie 30s, 50S, mRNA and polypeptide separates.
RNA of A-site forming peptide bond. This reaction is catalyzed by
RF-1 recognisaes UAA and UAg while RF-2 recognises UAA and UGA
peptidyltransferase.
while RF-3 dissociate 30S and 50S subunits.
Now, the t-RNA at P-site become uncharged
In case of eukaryotes only one release actor eRF causes dissociation.
Post translation modification:

The newly formed polypeptide may not be biologically functional so

it undergoes several folding and processing known as post
translation modification.

1. Amino terminal and carboxyl terminal modification: The N-

formylmethionine in case of bacteria is removed from polypeptide
chain and some carboxyl terminal are also removed by enzymatic
action to make functional protein. In case of eukaryotic protein,
amino terminal is N- acetylated.

2. Loss of signal sequences: In some protein the amino terminal end

is cleaved by specific peptidase so that protein loss its signaling
property.

3. Modification of individual aminoacids: The aminoacids may be

phosphorylated, acetylated for modification

4. Attachment of carbohydrate side chain: Carbohydrate side chain

is added to make protein functional. Eg, glycoprotein. Lipoprotein

5. Addition of isoprenyl group: In some protein, isoprenyl group is

added so to make protein active

Various protein factors involved in protein synthesis

Transcription in eukaryotes
Eukaryotic transcription is carried out in the nucleus of the cell by
one of three RNA polymerases, depending on the RNA being
transcribed, and proceeds in three sequential stages:
1. Initiation
2. Elongation
3. Termination.
The Three Eukaryotic RNA Polymerases (RNAPs)
The features of eukaryotic mRNA synthesis are markedly more
complex those of prokaryotes. Instead of a single polymerase
comprising five subunits, the eukaryotes have three polymerases
that are each made up of 10 subunits or more. Each eukaryotic
polymerase also requires a distinct set of transcription factors to
bring it to the DNA template.
RNA polymerase I is located in the nucleolus, a specialized nuclear
substructure in which ribosomal RNA (rRNA) is transcribed,
processed, and assembled into ribosomes. The rRNA molecules are
considered structural RNAs because they have a cellular role but are
not translated into protein. The rRNAs are components of the
ribosome and are essential to the process of translation. RNA
polymerase I synthesizes all of the rRNAs except for the 5S rRNA
molecule.
RNA polymerase II is located in the nucleus and synthesizes all
protein-coding nuclear pre-mRNAs. Eukaryotic pre-mRNAs undergo
extensive processing after transcription, but before translation. RNA
polymerase II is responsible for transcribing the overwhelming
majority of eukaryotic genes, including all of the protein-encoding
genes which ultimately are translated into proteins and genes for
several types of regulatory RNAs, including microRNAs (miRNAs)
and long-coding RNAs (lncRNAs).
RNA polymerase III is also located in the nucleus. This polymerase
transcribes a variety of structural RNAs that includes the 5S pre-
rRNA, transfer pre-RNAs (pre-tRNAs), and small nuclear pre-RNAs.
The tRNAs have a critical role in translation: they serve as the
adaptor molecules between the mRNA template and the growing
polypeptide chain. Small nuclear RNAs have a variety of functions,
including “splicing” pre-mRNAs and regulating transcription factors.
Not all miRNAs are transcribed by RNA Polymerase II, RNA
Polymerase III transcribes some of them.
Initiation of Transcription in Eukaryotes
Unlike the prokaryotic RNA polymerase that can bind to a DNA
template on its own, eukaryotes require several other proteins,
called transcription factors, to first bind to the promoter region and
then help recruit the appropriate polymerase. The completed
assembly of transcription factors and RNA polymerase bind to the
promoter, forming a transcription pre-initiation complex (PIC).
The most-extensively studied core promoter element in eukaryotes
is a short DNA sequence known as a TATA box, found 25-30 base
pairs upstream from the start site of transcription. Only about 10-
15% of mammalian genes contain TATA boxes, while the rest
contain other core promoter elements, but the mechanisms by
which transcription is initiated at promoters with TATA boxes is well
characterized.
The TATA box, as a core promoter element, is the binding site for a
transcription factor known as TATA-binding protein (TBP), which is
itself a subunit of another transcription factor: Transcription Factor
II D (TFIID). After TFIID binds to the TATA box via the TBP, five more
transcription factors and RNA polymerase combine around the
TATA box in a series of stages to form a pre-initiation complex. One
transcription factor, Transcription Factor II H (TFIIH), is involved in
separating opposing strands of double-stranded DNA to provide the
RNA Polymerase access to a single-stranded DNA template.
However, only a low, or basal, rate of transcription is driven by the
pre-initiation complex alone. Other proteins known as activators
and repressors, along with any associated coactivators or
corepressors, are responsible for modulating transcription rate.
Activator proteins increase the transcription rate, and repressor
proteins decrease the transcription rate.
Eukaryotic pre-mRNA processing
 When an RNA transcript is first made in a eukaryotic cell, it
is considered a pre-mRNA and must be processed into a
messenger RNA (mRNA).
 A 5' cap is added to the beginning of the RNA transcript, and
a 3' poly-A tail is added to the end.
 In splicing, some sections of the RNA transcript (introns) are
removed, and the remaining sections (exons) are stuck back
together.
 Some genes can be alternatively spliced, leading to the
production of different mature mRNA molecules from the
same initial transcript.

In bacteria, RNA transcripts are ready to act as messenger RNAs and

get translated into proteins right away. In eukaryotes, things are a
little more complex. The molecule that's directly made by
transcription is called a pre-mRNA, reflecting that it needs to go
through a few more steps to become an actual messenger RNA
(mRNA). These are:

 Addition of a 5' cap to the beginning of the RNA

 Addition of a poly-A tail (tail of A nucleotides) to the end of
the RNA
 Chopping out of introns, or "junk" sequences, and pasting
together of the remaining, good sequences (exons)

5' cap and poly-A tail

Both ends of a pre-mRNA are modified by the addition of chemical

groups. The group at the beginning (5' end) is called a cap, while the
group at the end (3' end) is called a tail. Both the cap and the tail
protect the transcript and help it get exported from the nucleus and
translated on the ribosomes (protein-making "machines") found in
the cytosol
The 5’ cap is added to the first nucleotide in the transcript during
transcription. The cap is a modified guanine (G) nucleotide, and it
protects the transcript from being broken down. It also helps the
ribosome attach to the mRNA and start reading it to make a protein.
When a sequence called a polyadenylation signal shows up in an
RNA molecule during transcription, an enzyme chops the RNA in
two at that site. Another enzyme adds about 100-200 adenine (A)
nucleotides to the cut end, forming a poly-A tail. The tail makes the
transcript more stable and helps it get exported from the nucleus to
the cytosol.
RNA splicing
The third big RNA processing event that happens is RNA splicing. In
RNA splicing, specific parts of the pre-mRNA, called introns are
recognized and removed by a protein-and-RNA complex called the
spliceosome. Introns can be viewed as "junk" sequences that must
be cut out so the "good parts version" of the RNA molecule can be
assembled.
What are the "good parts"? The pieces of the RNA that are not
chopped out are called exons. The exons are pasted together by the
spliceosome to make the final, mature mRNA that is shipped out of
the nucleus.
A key point here is that it's only the exons of a gene that encode a
protein. Not only do the introns not carry information to build a
protein, they actually have to be removed in order for the mRNA to
encode a protein with the right sequence. If the spliceosome fails to
remove an intron, an mRNA with extra "junk" in it will be made, and
a wrong protein will get produced during translation.
Splicing needs to precise and consistent. This careful cutting and
pasting is performed by the spliceosome, an enzyme complex made
of protein and small RNAs. Most introns contain marker sequences
at both of their ends, which are recognized by the small RNAs and
direct the spliceosome to remove the intron. Once the intron has
been cut out, the spliceosome will "glue" (ligate) the flanking exons
together.
Elongation in eukaryotes
RNA Polymerase II is a complex of 12 protein subunits. Specific
subunits within the protein allow RNA Polymerase II to act as its
own helicase, sliding clamp, single-stranded DNA binding protein, as
well as carry out other functions. Consequently, RNA Polymerase II
does not need as many accessory proteins to catalyze the synthesis
of new RNA strands during transcription elongation as DNA
Polymerase does to catalyze the synthesis of new DNA strands
during replication elongation.
However, RNA Polymerase II does need a large collection of
accessory proteins to initiate transcription at gene promoters, but
once the double-stranded DNA in the transcription start region has
been unwound, the RNA Polymerase II has been positioned at the
+1 initiation nucleotide, and has started catalyzing new RNA strand
synthesis, RNA Polymerase II clears or “escapes” the promoter
region and leaves most of the transcription initiation proteins
behind.
All RNA Polymerases travel along the template DNA strand in the 3’
to 5’ direction and catalyze the synthesis of new RNA strands in the
5’ to 3’ direction, adding new nucleotides to the 3’ end of the
growing RNA strand.
RNA Polymerases unwind the double stranded DNA ahead of them
and allow the unwound DNA behind them to rewind. As a result,
RNA strand synthesis occurs in a transcription bubble of about 25
unwound DNA basebairs. Only about 8 nucleotides of newly-
synthesized RNA remain basepaired to the template DNA. The rest
of the RNA molecules falls off the template to allow the DNA behind
it to rewind.
RNA Polymerases use the DNA strand below them as a template to
direct which nucleotide to add to the 3’ end of the growing RNA
strand at each point in the sequence. The RNA Polymerase travels
along the template DNA one nucleotide at at time. Whichever RNA
nucleotide is capable of basepairing to the template nucleotide
below the RNA Polymerase is the next nucleotide to be added. Once
the addition of a new nucleotide to the 3′ end of the growing strand
has been catalyzed, the RNA Polymerase moves to the next DNA
nucleotide on the template below it. This process continues until
transcription termination occurs.
Termination
The termination of transcription is different for the three different
eukaryotic RNA polymerases.
The ribosomal rRNA genes transcribed by RNA Polymerase I contain
a specific sequence of basepairs (11 bp long in humans; 18 bp in
mice) that is recognized by a termination protein called TTF-1
(Transcription Termination Factor for RNA Polymerase I.) This
protein binds the DNA at its recognition sequence and blocks
further transcription, causing the RNA Polymerase I to disengage
from the template DNA strand and to release its newly-synthesized
RNA.
The protein-encoding, structural RNA, and regulatory RNA genes
transcribed by RNA Polymerse II lack any specific signals or
sequences that direct RNA Polymerase II to terminate at specific
locations. RNA Polymerase II can continue to transcribe RNA
anywhere from a few bp to thousands of bp past the actual end of
the gene. However, the transcript is cleaved at an internal site
before RNA Polymerase II finishes transcribing. This releases the
upstream portion of the transcript, which will serve as the initial
RNA prior to further processing (the pre-mRNA in the case of
protein-encoding genes.) This cleavage site is considered the “end”
of the gene. The remainder of the transcript is digested by a 5′-
exonuclease (called Xrn2 in humans) while it is still being
transcribed by the RNA Polymerase II. When the 5′-exonulease
“catches up” to RNA Polymerase II by digesting away all the
overhanging RNA, it helps disengage the polymerase from its DNA
template strand, finally terminating that round of transcription.
In the case of protein-encoding genes, the cleavage site which
determines the “end” of the emerging pre-mRNA occurs between
an upstream AAUAAA sequence and a downstream GU-rich
sequence separated by about 40-60 nucleotides in the emerging
RNA. Once both of these sequences have been transcribed, a
protein called CPSF in humans binds the AAUAAA sequence and a
protein called CstF in humans binds the GU-rich sequence. These
two proteins form the base of a complicated protein complex that
forms in this region before CPSF cleaves the nascent pre-mRNA at a
site 10-30 nucleotides downstream from the AAUAAA site. The
Poly(A) Polymerase enzyme which catalyzes the addition of a 3′
poly-A tail on the pre-mRNA is part of the complex that forms with
CPSF and CstF.
Translation in eukaryotes
1. Site:

Translation occurs in the cytoplasm where the ribosomes are

located. Ribosomes are made of a small and large subunit which
surrounds the mRNA. In eukaryotic translation 80S ribosomes with
40S and 60S subunits are used. The mRNA is synthesized from DNA
only. In eukaryotes, there is single initiation and termination site.

2. Template:

This uses an mRNA sequence as a template to guide the synthesis of

a chain of amino acids that form a protein. Many types of
transcribed RNA, such as transfer RNA, ribosomal RNA, and small
nuclear RNA are not necessarily translated into an amino acid
sequence.

4. Factors Involved:

In eukaryotes, several factors are used in chain initiation such as (initialtion factor)
eIF2, eIF3, eIF4A, eIF4E, eIF4F and elF 4G. Two factors [EF-1 and EF-
2] are used in chain elongation. There is a single release factor RF
for recognition of three termination codons [UAA, UAG and UGA]. Initiation of Translation

5. Enzymes Involved: The initiation of translation in eukaryotes is complex, involving at

least ten eukaryotic initiation factors (eIFs). Some of the eIFs
In eukaryotes, two types of enzymes are used in translation. contain multiple (3-8) subunits. The process of translation initiation
Aminoacyl tRNA synthetase (an enzyme) catalyzes the bonding can be divided into four steps.
between specific tRNAs and the amino acids. The enzyme peptidyl
transferase connect A site and P site by forming a peptide bond [the Ribosomal dissociation.
nitrogen carbon bond] during elongation phase. (i) Formation of 43S preinitiation complex.
(ii) Formation of 48S initiation complex.
(iii) Formation of 80S initiation complex.
(iv) Ribosomal dissociation

The 80S ribosome dissociates to form 40S and 60S subunits.

Two initiating factors namely eIF3 and eIF-1A bind to the newly
formed 40S subunit, and thereby block its reassociation with 60S
subunit. For this reason, some workers name eIF-3 as anti-
association factor.
Formation of 43S preinitiation complex
A ternary complex containing met-tRNAi and eIF-2 bound to GTP
attaches to 40S ribosomal subunit to form 43S preinitiation
complex.
The presence of eIF-3 and eIF-1A stabilizes this complex (Note :
Met-tRNA is specifically involved in binding to the initiation condon
AUGs; hence the superscripti is used in mettRNAi).
Formation of 48S initiation complex
The binding of mRNA to 43S preinitiation complex results in the
formation of 48S initiation complex through the intermediate 43S
initiation complex. This, however, involves certain interactions
between some of the eIFs and activation of mRNA.
eIF-4F complex is formed by the association of eIF-4G, eIF-4A with
eIF-4E.
The so formed eIF-4F (referred to as cap binding protein) binds to
the cap of mRNA.
Then elF-4A and elF-4B bind to mRNA and reduce its complex
structure.
This mRNA is then transferred to 43S complex.
For the appropriate association of 43S preinitiation complex with
mRNA, energy has to be supplied by ATP.
Recognition of initiation codon:
The ribosomal initiation complex scans the mRNA for the
identification of appropriate initiation codon. 5c-AUG is the
initiation codon and its recognition is facilitated by a specific
sequence of nucleotides surrounding it.
This marker sequence for the identification of AUG is called as Kozak
consensus sequence.
In case of prokaryotes the recognition sequence of initiation codon
is referred to as Shine- Dalgarno sequence.
-Formation of 80S initiation complex
48S initiation complex binds to 60S ribosomal subunit to form 80S
initiation complex.
The binding involves the hydrolysis of GTP (bound to eIF-2). This
step is facilitated by the involvement of eIF-5.
As the 80S complex is formed, the initiation factors bound to 48S
initiation complex are released and recycled.
The activation of eIF-2 requires eIF-2B (also called as guanine
nucleotide exchange factor) and GTP.
The activated eIF-2 (i.e. bound to GTP) requires eIF2C to form the
ternary complex.
Regulation of initiation
The eIF-4F, a complex formed by the assembly of three initiation
factors controls initiation, and thus the translation process.
eIF4E, a component of eIF-4F is primarily responsible for the
recognition of mRNA cap. And this step is the rate-limiting in
translation.
eIF-2 which is involved in the formation of 43S preinitiation complex
also controls protein biosynthesis to some extent.
Elongation of Translation
Ribosomes elongate the polypeptide chain by a sequential addition
of amino acids. The amino acid sequence is determined by the order
of the codons in the specific mRNA. Elongation, a cyclic process
involving certain elongation factors (EFs), may be divided into three
steps.
(i) Binding of aminoacyl t-RNA to A-site.
(ii) Peptide bond formation.
(iii) Translocation.
Binding of aminoacyl—tRNA to A-site
The 80S initiation complex contains met-tRNAi in the P-site, and the
A-site is free.
Another aminoacyl-tRNA is placed in the A-site. This requires
proper codon recognition on the mRNA and the involvement of
elongation factor 1a (EF-Ia) and supply of energy by GTP.
As the aminoacyl-tRNA is placed in the A-site, EF-1D and GDP are
recycled to bring another aminoacyl-tRNA.
Peptide bond formation
The enzyme peptidyltransferase catalyses the formation of peptide
bond.
The activity of this enzyme lies on 28S RNA of 60S ribosomal
subunit. It is therefore the rRNA (and not protein) referred to as
ribozyme that catalyses the peptide bond formation.
As the amino acid in the aminoacyl-tRNA is already activated, no
additional energy is required for peptide bond formation. The net
result of peptide bond formation is the attachment of the growing
peptide chain to the tRNA in the A-site.
Translocation
As the peptide bond formation occurs, the ribosome moves to the
next codon of the mRNA (towards 3c-end). This process called
translocation, basically involves the movement of growing peptide
chain from A-site to P-site.
Translocation requires EF-2 and GTP.
GTP gets hydrolysed and supplies energy to move mRNA. EF-2 and
GTP complex recycles for translocation.
In recent years, another site namely exit site (E-site) has been
identified in eukaryotes. The deacylated tRNA moves into the E-site,
from where it leaves the ribosome.
In case of prokaryotes, the elongation factors are different, and they
are EF-Tu, EF-Ts (in place of of EF-1a) and EF-G (instead of EF-2).
Incorporation of amino acids
It is estimated that about six amino acids per second are
incorporated during the course of elongation of translation in
eukaryotes. In case of prokaryotes, as many as 20 amino acids can
be incorporated per second.Thus the process of
protein/polypeptide synthesis in translation occurs with great speed
and accuracy.
Termination of Translation
Termination is a simple process when compared to initiation and
elongation. After several cycles of elongation, incorporating amino
acids and the formation of the specific protein/ polypeptide
molecule, one of the stop or termination signals (UAA, UAG and
UCA) terminates the growing polypeptide.

The termination codons which act as stop signals do not have

specific tRNAs to bind. As theb termination codon occupies the
ribosomal A-site, the release factor namely eRF recognizes the stop
signal. eRF-GTP complex, in association with the enzyme
peptidyltransferase, cleaves the peptide bond between the
polypeptide and the tRNA occupying P-site. In this reaction, a water
molecule, instead of an amino acid is added. This hydrolysis releases
the protein and tRNA from the P-site. The 80S ribosome dissociates
to form 40S and 60S subunits which are recycled. The mRNA is also
released.

Eukaryotic initiation factor

Elongation factor for Prokaryote/Eukaryotic Translation  Removing the methionine- AUG code from the mature
chain.
 Adding a functional group to the amino acid or polypeptide
chain through phosphorylation, glycosylation, ubiquitination
and methylation etc.
 Forming disulfide bonds between cysteine residues
 Changes the function

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:

Post translational modification
Post-translational modifications, abbreviated s PTMs are the
biological modifications that occur after protein synthesis which
alters protein structure and function. Common PTMs are
phosphorylation, Methylation, acetylation, lipidation, hydroxylation
and ubiquitination. Post-translational modifications (PTMs) mainly
occur in the endoplasmic reticulum of the cell but sometimes
continue in the golgi bodies as well. A few common PTMs are
enlisted here,
 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.
 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.