TRANSLATION

(PROTEIN SYNTHESIS)
 Translation

• The translation of the mRNA codons into

amino acid sequences leads to the synthesis of
poypeptides, which then fold and/or aggregate
to form functional molecules called proteins.
•  The word “translation” is well-chosen because
the chemical language of nucleic acids (in
mRNA) is being changed into the chemical
language of polypeptides during the process.
 Cont------
• Proteins are the active participants in cell
structure and function.
• They are the “work horses” of the cell
• The main function of the genetic material is,
therefore, to encode the production of cellular
proteins in the correct cell, at the proper time,
and in suitable amounts
• Translation occurs in cytosol on ribosomes
 Cont------
• Note that the mRNA begins with a 5’
untranslated region
• In other words, the START codon is not at the
5’ end of the mRNA but somewhat
downstream (3’)
• Although it is not shown here, the STOP codon
is likewise not at the 3’ end of the mRNA.
There is a 3’ untranslated region after it.
 Basic requirements of translation
• mRNA to be translated
• tRNAs (at least 20)
• Ribosomes
• 20 aminoacids
• amino-acyl-tRNAsynthetases (at least 20)
• Energy in the form of ATP and GTP
• Enzymes and protein factors
 Cont------
mRNA
• It is a template for protein synthesis
• Eukaryotic mRNA is monocystronic meaning
that there is only one coding sequence on each
mRNA producing only one polypeptide chain
• Prokaryotic mRNA is polycistronic, often
encoding for more than one polypeptide on the
same mRNA.
 Cont------
tRNA
• It is the adaptor molecule in protein synthesis
• In the 1950s, Francis Crick and Mahon Hoagland
proposed the adaptor hypothesis, which
hypothesized that tRNAs play a direct role in
the recognition of codons in the mRNA.
• In particular, the hypothesis proposed that
tRNA has two functions:
 Cont------
1. Recognizing a 3-base codon in mRNA
2. Carrying an amino acid that is specific for
that codon to the translation machinery
• During mRNA-tRNA recognition, the anticodon
in tRNA binds to a complementary codon in
mRNA:
 
 Modified Nucleotides and the Wobble Hyothesis
 

• In addition to the normal A, U, G and C

nucleotides, tRNAs commonly contain modified
nucleotides
• More than 60 of these can occur
•  As mentioned earlier, the genetic code is
degenerate
• With the exception of serine, arginine and
leucine, this degeneracy always occurs at the
codon’s third position
 Cont------
– To explain this pattern of degeneracy, Francis Crick
proposed in 1966 the wobble hypothesis
– In the codon-anticodon recognition process, the first
two positions pair strictly according to the A – U /G –
C rule
– However, the third position can actually “wobble” or
move a bit, thus tolerating certain types of
mismatches
• The modified nucleotides in the tRNA anticodon
allow this “wobbling” to occur.
 Cont------
• The anticodon of tRNA identifies a number of
synonym codons of one amino acid that differ
at the 3rd base.
• Examples are the two arginine codons, AGA
and AGG bind same UCU anticodon and the
three glycine codons, GGU, GGC and GGA bind
same CCI anticodon.
 The Ribosome
•  Translation occurs on the surface of a large
macromolecular complex termed the
ribosome
• A ribosome is composed of structures called
the large and small subunits
• Each subunit is formed from the assembly of
• Proteins
• Ribosomal RNA (rRNA)
 Cont------
• Ribosomes contain 3 discrete sites:
–Peptidyl site (P site)
–Aminoacyl site (A site)
–Exit site (E site)
 Stages of translation
• The process of protein synthesis includes four
steps:
• I. Activation of amino acids.
• II. Initiation.
• III. Elongation.
• IV. Termination.
 1. Activation of amino acids:
• It is catalyzed by amino-acyl-tRNAsynthetase that
is specialized in sticking a specific amino acid to a
specific tRNA, as follows,
• Amino acid + ATP  Amino-acyl-AMP + PPi 2Pi
• Amino-acyl-AMP + tRNA Amino-acyl-tRNA +
AMP
• The -COOH of the amino acid binds to the 3'-OH
group of adenine of the acceptor arm of the tRNA
(i.e., 3'-ACC).
 2. Initiation:
• Initiation of protein synthesis requires
identification of mRNA for translation by
ribosomes. Requirements of the initiation
step are:
• a) Amino-acyl-tRNA. b) Ribosome. c) mRNA.
d) GTP and ATP.
• e) At least 10 eukaryotic initiation factors
(eIFs).
 A. Formation of the 40S preinitiation
complex:
• Eukaryotic initiation factor-1 (eIF1) dissociates
the complete ribosome into its 40S and 60S
subunits. Then, initiation factor-3 (eIF-3)
prevents reassociation these subunits.
• The amino-acyl-tRNA (met-tRNA) interacts
with initiation factor-2 (eIF-2) bound to GTP
that enables it to bind the 40S-eIF-1-eIF3
complex to give the 40S preinitiation complex.
B. Formation of the 40S initiation complex:

• Initiation factor-4 (eIF-4) binds the cap of

mRNA and activates it to bind the 40S
preinitiation complex to form the 40S initiation
complex with hydrolysis of ATP into ADP + Pi.
• This complex scans mRNA for the initiation AUG
that is the 5'-most AUG with a specific
sequence around it.
 Cont------
• Thus, anticodon of amino-acyl-tRNA is brought
into contact with the translation initiation
codon in mRNA.
• The newly synthesized protein always starts
with methionine (formyl-methionine in
prokaryote) as the N-terminal amino acid as
translation always begins at AUG.
C. Formation of the 80S initiation complex:

• Initiation factor-5 (eIF5) hydrolyzes GTP on eIF2

into GDP + Pi and activates binding of the 40S
initiation complex to 60S ribosomal subunit
with release of initiation factors-1, -2, -3 and -4.
• The complete ribosome contains two amino-
acyl-tRNA-binding sites. These are the P site
(peptidyl site, i.e., preceding amino-acyl-tRNA
site carrying one or more amino acids) and the
A site (i.e., following amino-acyl-tRNA site).
 Cont------
• The first amino-acyl-tRNA carrying the first amino
acid in the polypeptide chain will be automatically
located at P site and the next amino-acyl-tRNA
enters at A site, see the figure below.
• When cell is under stress conditions that makes it
unable to synthesize protein, e.g., lake of amino
acids or glucose, growth factor deprivation, or heat
shock, eIF2 undergoes inhibitory phosphorylation by
specific kinases to prevent formation of 40S
preinitiation complex and hence protein synthesis.
 3. Elongation:

• An elongation factor-1 (eEF-1) binds with amino-

acyl-tRNA with hydrolysis of GTP into GDP + Pi,
forming a complex.
• This complex allows amino-acyl-tRNA to enter at A
site of ribosome with the release of eEF-1.
• The free -NH2 group of the new amino acid binds
the -COOH group of the first amino acid (Met) with
the transfer of whole peptide chain to tRNA at A site
by peptidyltransferase with the release of the free
tRNA at P site
 Cont------
• Elongation factor 2 (eEF-2, translocase) translocates
the whole complex with the newly formed peptidyl-
tRNA a one-codon distance along the mRNA in 5' to
3' direction.
• Translocation requires hydrolysis of GTP into GDP + Pi
and creates a new free A site for entrance of a new
amino-acyl-tRNA to recognize a new codon and so
on.
• Polypeptide chain is thus increased by one amino
acid each time.
 4. Termination:

• When a termination codon in mRNA appears

at A-site, there is no tRNA that can recognize it,
but rather, releasing factors (eRFs) recognize it.
• A releasing factor activates the
peptidyltransferase to hydrolyze and release
the peptide chain on tRNA at P site to free
tRNA then mRNA and 80S ribosome
dissociates into its subunits.
Post-translation Modification of Proteins:

• Activation by hydrolysis of an extra-peptide

(pre-pro-proteins) or zymogens.
• Glycosylation that occurs in endoplasmic
reticulum and Golgi apparatus.
• Hydroxylation of proline and lysine, e.g., in
collagen.
• Phosphorylation of tyrosine, serine or
threonine.
 Polypeptide Localization
•  The amino acid sequences of newly-
synthesized polypeptides contain “sorting
signals” that tell the cell where they belong
•  These are especially important in eukaryotes,
where each sorting signal is recognized by a
specific cellular component
• These cellular components facilitate the sorting
of the protein to its correct compartment
 Effect of antibiotics on protein synthesis:

• Many antibiotics selectively inhibit protein

synthesis in bacterial because of the distinction
between eukaryotic and prokaryotic ribosomal
system.
• Aminoglycosides (streptomycin, gentamycin
and amikin), they binds the 30S rRNA and
disturb mRNA binding to the ribosome.
• Tetracyclines prevent the binding of amino-
acyl-tRNA to A site.
 Cont------
• Chloramphenicol inhibits peptidyltransferase.
• Puromycin has a structure similarity with tyrosyl-
tRNA and cause premature release of the peptide
in both eukaryotes and prokaryotes.
• Cycloheximide inhibits peptidyltransferase in
eukaryotes only.
• Diphtheria exotoxin ADP-ribosylates eEF2 to
inhibit its function.
• Erythromycin Inhibits translocation
 Cont------
• Many polypeptides will not fold properly and become
functional proteins until they are properly localized
within the cell
• Sometimes, mutations lead to changes in the amino
acid sequence of the polypeptide that prevent its
localization
• Such polypeptides are eventually degraded
• Localization mutations are usually null mutations (the
protein coded by the gene has no residual function in
the cell)
 Cont------
Polyribosomes (Polyosomes)
• Multiple ribosomes translate one mRNA
simultaneously, one after the other. This
phenomenon is called polyribosome.
 Regulation of gene expression

• Gene expression is controlled at:

• A. At transcription level.
• B. At post transcription level.
 A. Transcriptional control of gene
expression:
• Each differentiated cell has the capacity to
synthesize certain types of protein in suitable
amounts through differential regulation of
gene expression.
• Regulation of gene expression can be
illustrated by the Lac-Operon model in E. Coli
as the simplest model for regulation of gene
expression since regulation of other genes in E.
Coli or eukaryotes are far more complex.
 Cont------
• Operon is a coordinated unit of gene
expression (Lac = lactose-metabolizing
enzymes, Oper = operation, and On = is on).
• Lac-Operon is formed of two genes with their
regulatory sequences. One gene is
constitutively regulated regulatory gene, the
LacI-gene, i.e., Lac-gene inhibitory gene that
gives a monocistronic mRNA translated into
the repressor protein.
 Cont------
• The other gene is structural, Lac-gene formed
of three units (Z, Y and A) and gives a
polycistronic mRNA translated into three
proteins (-galactosidase, lactose permease
and acetylase).
• E. Coli can metabolize glucose, glycerol,
lactose or galactose as a source of energy with
preference to glucose.
 Cont------
• For the integrated metabolism of lactose the
bacterium requires lactose permease that permits
entry of lactose into the cells
• -galactosidase that hydrolyzes lactose into glucose
and galactose.
• They are co-regulated since they are produced as
one polycistronic mRNA along with a third enzyme
of unknown function, i.e., acetylase(it may acetylate
specific proteins and facilitate gene induction).
 Cont------
• Since Lac-gene is an inducible gene, in
presence of glucose the bacterium contains a
few molecules of these enzymes per cell.
• But when the media lacks glucose, the
bacterium expresses this gene at a very high
rate (as much as 100-fold the uninduced rate)
producing large amount of these enzymes per
cell to increase lactose metabolism.
 Promoter (RNA polymerase binding site)
CAP binding site Operator (lac repressor binding site)
Promoter lacZ lacY lacA
lacI
DNA

lac Operon
 Cont------
• Jacob and Monod elucidated the mechanism
of the coordinated regulation of the expression
(induction/repression) of that Operon in 1961.
• As above, the Lac-gene has a promoter
sequence downstream of which there is the
Operator sequence at which Lac repressor
binds to hinder the binding of RNA polymerase
to the promoter,
 Cont------
• and upstream of which there is the CAP sequence at
which the catabolite-activated DNA-binding protein
(CAP, cAMP-dependent) binds to activate binding of
RNA polymerase.
• The regulatory Lacrepressor DNA-binding protein is
the product of Lac repressor gene (Lac-I gene) which
has the promoter as the only regulatory sequence
and is a regulatory gene with a constitutive constant
rate of transcription and hence protein synthesis.
The two states of Lac-Operon (Repression and induction):

1. Repression:
• When E. Coli is grown in presence of glucose,
transcription of the Lac-gene is repressed.
• Repression is mediated by Lac repressor, which binds
as a tetramer to the operator sequence preventing
the binding of RNA polymerase to the promoter and
prevents the transcription of the Lac-gene.
• The repressor is a negative regulator, and its
sequence is a silencer on expression of the Operon
 2.Induction (or derepression):
• When glucose is absent, the Lac-gene is
induced, i.e., expression rate of the Operon is
increased.
• Starvation of the bacterium of glucose leads
to increase of cAMP that binds and activates
the DNA binding of the Catabolite gene
Activator Protein (CAP).
 Cont------
• The CAP binds to the CAP binding sequence of
DNA up-stream the promoter to facilitate
binding of RNA polymerase to the promoter to
induce transcription of the Lac-gene at a low
level.
• Therefore, cAMP-activated CAP is a positive
regulator, and its sequence is an enhancer.
 Cont------
• The produced small amount of permease
facilitates entrance of lactose into the cell.
• Lactose (an inducer) has high affinity to bind
the repressor causing a change in its
conformation. This change prevents the
repressor from binding to the operator.
• Therefore, lactose acts as an inducer and the
operator becomes free for higher rate of
transcription by RNA polymerase.
B. Post-Transcriptional control of gene expression:

• Gene expression is also regulated at the levels

of RNA processing (e.g., capping, tailing,
alternative splicing and editing), RNA
transport, mRNA half-life and rate of
translation (e.g., proteins utilized in iron
absorption, transport and storage).