TRANSLATION

(PROTEIN SYNTHESIS)
 Translation

• The translation of the mRNA codons into

amino acid sequences leads to the synthesis of
polypeptides, 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 on
mRNA:
 Modified Nucleotides and the Wobble Hypothesis

• 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 bacteria 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.