TRANSLATION AND GENE EXPRESSION

The protein synthesis which involves the translation of nucleotide base sequence of mRNA
may be divided into the following stages

I. Requirement of the components

II. Activation of amino acids
III. Protein synthesis proper
IV. Chaperones and protein folding
V. Post-translational modifications.
I. REQUIREMENT OF THE
COMPONENTS
1. Amino acids : Of the 20 amino acids found in protein structure, half of them (10) can
be
synthesized by man. About 10 essential amino acids have to be provided through the diet. If
there is a deficiency in the dietary supply of any one of the essential amino acids, the
translation stops. It is, therefore, necessary that a regular dietary supply of essential amino
acids, in sufficient quantities, is maintained, as it is a prerequisite for protein synthesis.

2. Ribosomes : The functionally active ribosomes are the centres or factories for protein
synthesis. Each ribosome consists of two subunits—one big and one small. The functional
ribosome has two sites—A site and P site. Each site covers both the subunits. A site is for
binding of aminoacyl tRNA and P site is for binding peptidyl tRNA, during the course of
translation. Some authors consider A site as acceptor site, and P site as donor site. In case of
eukaryotes, there is another site called exit site or E site. Thus, eukaryotes contain three sites
(A, P and E) on the ribosomes.

3. Messenger RNA (mRNA) : The specific information required for the synthesis of a given
protein is present on the mRNA. The DNA has passed on the genetic information in the form
of codons to mRNA to translate into a protein sequence.

4. Transfer RNAs (tRNAs) : They carry the amino acids, and hand them over to the growing
peptide chain. The amino acid is covalently bound to tRNA at the 3’-end. Each tRNA has a
three nucleotide base sequence—the anticodon, which is responsible to recognize the codon
(complementary bases) of mRNA for protein synthesis.

5. Energy sources : Both ATP and GTP are required for the supply of energy in protein
synthesis.

6. Protein factors : The process of translation involves a number of protein factors. These
are
needed for initiation, elongation and termination of protein synthesis. The protein factors are
more complex in eukaryotes compared to prokaryotes.

II. ACTIVATION OF AMINO ACIDS Amino acids are activated and attached to tRNAs in
a two step reaction. A group of enzymes—namely aminoacyl tRNA synthetases— are
required for this process. The amino acid is first attached to the enzyme utilizing ATP to
form enzyme-AMP-amino acid complex. The amino acid is then transferred to the 3’ end of
the tRNA to form aminoacyl tRNA.

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III. PROTEIN SYNTHESIS PROPER

The protein or polypeptide synthesis occurs on the ribosomes (rather polyribosomes). The
mRNA is read in the 5’ 3’ direction. Translation proper is divided into three stages
initiation, elongation and termination (as it is done for transcription).

INITIATION OF TRANSLATION
The process of translation initiation can be divided into four steps

1. Ribosomal dissociation.
2. Formation of 43S preinitiation complex.
3. Formation of 48S initiation complex.
4. Formation of 80S initiation complex.

Ribosomal dissociation
The 80S ribosome dissociates to form 40S and 60S subunits. Two initiating factors namely
eIF- 3 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

Formation of 48S initiation complex

The binding of mRNA to 43S preinitiation complex results in the formation of 48S initiation
Complex. 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.

Recognition of initiation codon : The ribosomal initiation complex scans the mRNA for the
identification of appropriate initiation codon. 5’-AUG is the initiation codon and its

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recognition is facilitated by a specific sequence of nucleotides surrounding it. This marker

sequence for the identification of AUG is called as Kozak consensus sequences. 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.
Regulation of initiation
The eIF-4F, a complex formed by the assembly of three initiation factors controls initiation,
and thus the translation process. eIF- 4E, 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.

Initiation of translation in prokaryotes- The 30S ribosomal subunit is bound to initiation

factor 3 (IF-3) and attached to ternary complex of IF-2, formyl mettRNA and GTP. Another
initiation factor namely IF-I also participates in the formation of preinitiation complex. The
recognition o initiation codon AUG is done through Shine- Dalgarno sequence. A 50S
ribosome unit is now bound with the 30S unit to produce 70S initiation complex in
prokaryotes.

ELONGATION OF TRANSLATION
Elongation, a cyclic process involving certain elongation factors (EFs), may be divided into
three steps
1. Binding of aminoacyl t-RNA to A-site.
2. Peptide bond formation.
3. Translocation.

Binding of aminoacyl—tRNA to A-site

The 80S initiation complex contains mettRNAi 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-1a 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. 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.
This process called translocation, basically involves the movement of growing peptide chain
from A-site to P-site. Translocation requires EF-2 and GTP. Another site namely exit site (E-
site) has been identified in eukaryotes. The deacylated tRNA moves into the E-site, from

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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).

TERMINATION OF TRANSLATION
After several cycles of elongation, incorporating aminoacids 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 the termination codon occupies the ribosomal A-site, the
release factor namely eRF recognizes the stop signal. eRF-GTP complex, in association with

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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.

GENE REGULATION—GENERAL
The regulation of the expression of genes is absolutely essential for the growth,
development, differentiation. There are two types of gene regulation-positive and negative.
1. Positive regulation : The gene regulation is said to be positive when its expression is
increased by a regulatory element (positive regulator).
2. Negative regulation : A decrease in the gene expression due to the presence of a
regulatory element (negative regulator) is referred to as negative regulation.

Constitutive and inducible genes

The genes are generally considered under two categories
1. Constitutive genes : The products (proteins) of these genes are required all the time in a
cell. Therefore, the constitutive genes (or housekeeping genes) are expressed at more or less
constant rate in almost all the cells and, further, they are not subjected to regulation e.g.
the enzymes of citric acid cycle.

2. Inducible genes : The concentration of the proteins synthesized by inducible genes is

regulated by various molecular signals. An inducer increases the expression of these genes
while a repressor decreases, e.g. tryptophan pyrrolase of liver is induced by tryptophan.

THE OPERON CONCEPT

The operon is based on the regulation of lactose metabolism in E. coli. This is popularly
known as lac operon.
LACTOSE (LAC) OPERON- Structure of lac operon
The lac operon consists of a regulatory gene (I; I for inhibition), operator gene (O) and three
structural genes (Z, Y, A). Besides these genes, there is a promoter site (P), next to the
operator gene, where the enzyme RNA polymerase binds. The structural genes Z, Y and A
respectively, code for the enzymes -galactosidase, galactoside permease and galactoside
acetylase. -Galactosidase hydrolyses lactose (-galactoside) to galactose and glucose while
permease is responsible for the transport of lactose into the cell. The function of acetylase
(coded by A gene) remains a mystery. The structural genes Z, Y and A transcribe into a
single large mRNA with 3 independent translation units for the synthesis of 3 distinct
enzymes. An mRNA coding for more than one protein is known as polycistronic mRNA.

Repression of lac operon

The regulatory gene (I) is constitutive. It is expressed at a constant rate leading to the
synthesis of lac repressor. Lac repressor is a tetrameric (4 subunits) which specifically binds
to the operator gene (O). This prevents the binding of the enzyme RNA polymerase to the
promoter
site (P), thereby blocking the transcription of structural genes (Z, Y and A). This is what
happens in the absence of lactose in E. coli.

Derepression of lac operon

In the presence of lactose (inducer) in the medium, a small amount of it can enter the E. coli
cells. The repressor molecules have a high affinity for lactose. The lactose molecules bind

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and induce a conformational change in the repressor. The result is that the repressor gets
inactivated and, therefore, cannot bind to the operator gene (O). The RNA polymerase
attaches to the DNA at the promoter site and transcription proceeds, leading to the formation
of polycistronic mRNA (for genes Z, Y and A) and, finally, the 3 enzymes. Thus, lactose
induces the synthesis of the three enzymes -galactosidase, galactoside permease and
galactoside acetylase. Lactose acts by inactivating the repressor molecules, hence this
process is known as derepression of lac operon.

TRYPTOPHAN OPERON Tryptophan is an aromatic amino acid, and is required for the
synthesis of all proteins that contain tryptophan. If tryptophan is not present in the medium in

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adequate quantity, the bacterial cell has to make it, as it is required for the growth of the
bacteria.
This operon contains five structural genes (trpE, trpD, trpC, trpB, trpA), and the regulatory
elements—primary promoter (trpP), operator (trpO), attenuator (trpa), secondary internal
promoter (TrpP2), and terminator (trpt). The five structural genes of tryptophan operon code
for three enzymes (two enzymes contain two different subunits) required for the synthesis
of tryptophan from chorismate. The tryptophan repressor is always turned on. Thus lactose
operon is inducible, whereas tryptophan operon is repressible.

Tryptophan operon regulation by a repressor

Tryptophan acts as a corepressor to shut down the synthesis of enzymes from tryptophan
operon. This is brought out in association with a specific protein, namely tryptophan
repressorTryptophan repressor, a homodimer (contains two identical subunits) binds with
two molecules of tryptophan, and then binds to the trp operator to turn off the transcription.
Two polycistronic mRNAs are produced from tryptophan operon—one derived from all the
five structural genes, and the other obtained from the last three genes.

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