TRANSLATION

Prepared by
A. Nandakumar, M.Tech
TRANSLATION
 The process involves in converting the nucleic
acid “language,” the genetic code, to protein
“language,” and is therefore known as
translation.
 This was first formulated by Sir Francis Crick.
AMINO ACID CODING
 There are 20 amino acids in proteins but only
four different bases in the mRNA.
 During translation,the bases of mRNA are read
off in groups of three, which are known as
codons.
 Four different bases gives 64 possible groups of
three bases; that is, 64 different codons in the
genetic code.
 In addition, three of the codons are used for
punctuation. Those are the stop codons that
signal the end of a polypeptide chain.
 To read the codons,a set of adapter molecules that
recognize the codon on the mRNA at one end and
carry the corresponding amino acid attached to
their other end is needed.
 These adapters are small RNA molecules, or
transfer RNA (tRNA).
 This adapter contains anticodon at one end which
are complementary to the three bases of the
codon on the mRNA.
 The codon and anticodon recognize each other by
base pairing and are held together by hydrogen
bonds.
PROKARYOTES
 70s Ribosome composed of 30s
(small subunit) & 50s (large subunit).
 Small subunit - 16s rRNA contains
21 different proteins.
 Large subunit – 5s & 23s rRNA
contains 32 different proteins.
 The initiator tRNA is first charged
with methionine by methionyl-tRNA
synthetase. The methionine residue is
then converted to N-
formylmethionine by transformylase.
 Multiple start sites.
 Initiation factors, IF1, IF2 and IF3.
COMPONENTS OF RIBOSOME & DIFFERENCE
EUKARYOTES
 80s Ribosome composed of 60s
(large subunit) & 40s (small subunit).
 5’ r cap instead of Shine-Dalgarno
sequence.
 In eukaryotes, the methionine on the
initiator tRNA is not modified.
 The initiating codon in eukaryotes is
always AUG.
 Initiation factors, eIF1, eIF2, eIF3,
eIF4, eIF5 and eIF6.
OVERVIEW OF PROKARYOTIC TRANSLATION
TRANSLATION: THE PROCESS OF PROTEIN
SYNTHESIS
1. Ribosomes translate the genetic message of mRNA into
proteins.
2. The mRNA is translated 5’3’, producing a corresponding
N-terminal  C-terminal polypeptide.
3. Amino acids bound to tRNAs are inserted in the proper
sequence due to:
a. Specific binding of each amino acid to its tRNA.
b. Specific base pairing between the mRNA codon and tRNA
anticodon.
CHARGING tRNA
1. Aminoacyl-tRNA synthetase attaches amino acids to their
specific tRNA molecules. The charging process
(aminoacylation) produces a charged tRNA (aminoacyl-
tRNA), using energy from ATP hydrolysis.
2. There are 20 different aminoacyl-tRNA synthetase enzymes,
one for each amino acid. Some of these enzymes recognize
tRNAs by their anticodon regions.
3. The amino acid and ATP bind to the specific aminoacyl-
tRNA synthetase enzyme. ATP loses two phosphates and the
resulting AMP is bound to the amino acid, forming
aminoacyl-AMP.
4. The tRNA binds to the enzyme, and the amino acid is
transferred onto it, displacing the AMP. The aminoacyl-tRNA
is released from the enzyme.
 CHARGING OF A tRNA MOLECULE BY AMINOACYL-
tRNA SYNTHETASE
5. The amino acid is now covalently attached by its carboxyl
group to the 3’r end of the tRNA. Every tRNA has a 3’r
adenine, and the amino acid is attached to the 3’r–OH or 2’r–
OH of this nucleotide.
CHARGED tRNA
FORMYLATION OF tRNA
INITIATION
 In prokaryotes, initiation requires the large and small ribosome
subunits, the mRNA, the initiator tRNA, three initiation factor(IFs)
and GTP.
 IF1 and IF3 bind to the 30S subunit and prevent the large subunit
binding.
 IF2+GTP can then bind and will help the initiator tRNA to bind later.
 This small subunit complex can now attach to an mRNA via its
ribosome-binding site.
 The initiator tRNA can then base-pair with the AUG initiation codon
which releases IF3 thus creating the 30S initiation complex.
 The large subunit then binds, displacing IF1 and IF2+GTP, giving the
70S initiation comple which is the fully assembled ribosome at the
correct position on the mRNA.
SHINE-DALGARNO SEQUENCE
 Shine-Dalgarno sequence is an ribosomal binding site in
prokaryotic mRNA, generally located around 8 bases
upstream of the start codon AUG.
 Shine-Dalgarno sequence exists both in bacteria &
archaea and also in some chloroplast & mitochondria.
 Shine-Dalgarno sequence helps to make ribosome
available to the mRNA to initiate protein synthesis by
aligning it with the start codon.
ELONGATION
 Elongation involves the three factors(Efs), EF-Tu, EF-Ts and EF -
G, GTP, charged tRNA and the 70S initiation complex(or its
equivalent). It takes place in three steps.
 A charged tRNA is delivered as a complex with EF-Tu and GTP.
The GTP is hydrolyzed and EF-Tu.GTP is released which can be
re-used with the help of EF-Ts and GTP(via the EF-Tu-EF-Ts
exchange cycle).
 Peptidyl transferase makes a peptide bond by joining the two
adjacent amino acid without the input of more energy.
 Translaocase(EF-G), with energy from GTP, move the ribosome
one codon along the mRNA, ejecting the uncharged tRNA and
transferring the growing peptide chain to the P-site.
ACTION OF PEPTIDYL TRANSFERASE
 The two aminoacyl-tRNAs are positioned by the ribosome
for peptide bond formation, which occurs in two steps:
a. In the P site, the bond between the amino acid and its
tRNA is cleaved.
b.Peptidyl transferase forms a peptide bond between the
now-free amino acid in the P site and the amino acid
attached to the tRNA in the A site. Experiments indicate
that the 23S rRNA is most likely the catalyst for peptide
bond formation.
c. The tRNA in the A site now has the growing
polypeptide chain attached to it.
TERMINATION
 Release factors(RF1 or RF2) recognize the stop codon
and, helped by RF3, make peptidyl transferase join the
polypeptide chain to a water molecule, thus releasing it.
 Ribosome release factor helps to dissociate the ribosome
subunit from the mRNA.
TRANSLATION FACTORS
OVERVIEW OF EUKARYOTIC TRANSLATION
INITIATION
 This is the major point of difference between prokaryotic
and eukaryotic protein synthesis, there being at least nine
eIF involved.
 Functionally, these factors can be grouped. They either
bind to the ribosome subunit or to the mRNA, deliver the
initiator tRNA or displace other factors.
 In contrast to the events in prokaryotes, initiation involves
the initiator tRNA binding to the 40S subunit before it can
bind to the mRNA.
 Phosphorylation of eIF2, which delivers the initiator
tRNA, is an important control point.
ELONGATION & TERMINATION
 This stage of protein synthesis is essentially identical to
that described for prokaryotes.
 The factors EF-Tu, EF-Ts and equivalents called eEF1α,
eEF1βγ and eEF2 respectively, which carry out the same
roles. EF-G have direct eukaryotic activity.
 Eukaryotes use only one release factor (eRF), which
requires GTP, for termination of protein synthesis. It can
recognize all three codons.
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