Translation (Protein Synthesis)

 Translation is the process by which linear sequence of nucleotides or

codons in a molecule of mRNA directs the specific linear sequence
of amino acids in a polypeptide.
 Protein synthesis occurs over the ribosomes.
 Ribosomes, therefore, are also called protein factories.
 The mRNA translates the coded information by bringing amino acids
in a particular sequence during polypeptide synthesis through the
agency of tRNAs.

Machinery for Protein Synthesis

1. Ribosomes
 Sites of protein synthesis
 Each ribosome has two unequal parts, small and large.
 The larger subunit of ribosome has a groove for pushing out the
newly formed polypeptide and protecting the same from cellular
enzymes.
 The smaller subunit fits over the larger one like a cap but leaves a
tunnel for mRNA.
 The two subunits come together only at the time of protein formation.
The phenomenon is called association. Mg2+ is essential for it.
 Soon after the completion of protein synthesis, the subunits
separate. The phenomenon is called dissociation.
 Ribosomes usually form rosette or helical groups during active
protein synthesis. They are known as polyribosomes or
polysomes.
 The different ribosomes of a polysome are held together by a strand
of messenger RNA.
 Polyribosome helps to produce a number of copies of the same
polypeptide.
 The adjacent ribosomes of a polyribosome are about 34 nm apart.
 The different parts of a ribosome connected with protein synthesis
are:
(i) A tunnel for mRNA. It lies between the two subunits.
(ii) A groove for passage of newly synthesised polypeptide. The groove
is part of the larger subunit.
(iii) There are three reactive sites- P, A and E. P-site (peptidyl transfer
or donor site) is jointly contributed by the two ribosomal subunits. А-
site (amino-acyl or acceptor site) is situated on the larger subunit of
ribosome. It faces the tunnel between the two subunits. E or exit site is
part of larger subunit facing the tunnel site.
(iv) Enzyme peptidyl transferase is a ribozyme. It is component of
larger subunit of ribosome.
(v) Smaller subunit of ribosome has a point for recognizing mRNA and
binding area for initiation factors.
2. Amino Acids
 Hundreds of different types of proteins may be manufactured in a
single cell.
 All types of proteins are formed from the same amino acids.
 It is the arrangement of amino acids in the polypeptides and the
number of the latter which provide specificity to the proteins.
 There are some 20 amino acids and amides which constitute
building blocks or monomers of proteins.
 They occur in the cellular pool.
3. mRNA
 It is messenger RNA which brings coded information from DNA and
takes part in its translation by bringing amino acids in a particular
sequence during the synthesis of polypeptide.
 However, the codons of mRNA are not recognized by amino acids
but by anticodons of tRNAs.
4. tRNAs
 They are transfer or soluble RNAs which pick up particular amino
acids (at CCA or 3´ end) in the process called charging.
 The charged tRNAs take the same amino acid to mRNA over
particular codons corresponding to their anticodons.
 A tRNA can pick up only a specific amino acid though an amino acid
can be specified by 2-6 tRNAs.
 Each tRNA has an area for coming in contact with ribosome (TΨC)
and the enzyme amino acyl tRNA synthetase (DHU).
5. Amino-Acyl-tRNA-Synthetase
 It is the enzyme that helps in combining amino acid to its particular
tRNA.
 The enzyme is specific for each amino acid.
 It is also called aa-activating enzyme.

Mechanism of Protein Synthesis

1. Activation of Amino Acids
 It is carried out by activating enzymes, known as aminoacyl tRNA
synthetases.
 In the presence of ATP, an amino acid combines with its specific
aminoacyl-tRNA synthetase.
 Mg2+ is required.
 It produces amino-acyl-adenylate-enzyme complex.
 The energy made available to amino acid during its activation is later
used in formation of peptide bonds.
2. Synthesis of Aminoacyl tRNAs
 The amino-acyl-adenylate-enzyme complex reacts with tRNA
specific for the amino acid to form aminoacyl-tRNA complex.
 Enzyme and AMP are released.
 tRNA complexed with amino acid is sometimes called charged tRNA.

3. Initiation
 It requires factors called initiation factors.
 There are three initiation factors in prokaryotes- IF3, IF2 and IF1.
 Eukaryotes have nine initiation factors- eIF2, eIF3, eIF1, eIF4A,
eIF4B, eIF4C, eIF4D, eIF5, eIF6.
 Out of these IF3 or eIF2 is attached to smaller subunit of ribosome in
the dissociated state.
 mRNA attaches itself to smaller subunit of ribosome.
 Leader region of mRNA has 8-13 nucleotide long sequence called
Shine-Dalgarno sequence in prokaryotes and Kozak sequence in
eukaryotes.
 The nucleotides of Shine-Dalgarno sequence or Kozak sequence are
complementary to the nucleotides present at the 3′ end of rRNA.
 The attachment is such that initiation codon of mRNA (AUG or GUG)
comes to lie at P-site.
 Initiation factor already present in smaller subunit catalyzes the
reaction (eIF2 in eukaryotes and IF3 in prokaryotes).

 Aminoacyl tRNA complex specific for the initiation codon

(methionine-tRNA or valine-tRNA) reaches the P-site.
 Anticodon (e.g., UAC of tRNAMet) establishes temporary hydrogen
bonds with the initiation codon (e.g., AUG) of mRNA.
 The codon-anticodon reaction occurs in the presence of initiation
factor eIF3 in eukaryotes and IF2 in prokaryotes.
 The step also requires energy which is provided by GTP.
 The initiating methionine accepting tRNA is charged with non-
formylated methionine (tRNA mMet) in the cytoplasm of eukaryotes
and formylated methionine (tRNA fMet) in prokaryotes, plastids and
mitochondria. tRNA engaged in transferring formylated methionine is
different than the one that transfers nonformylated methionine.
 In the presence of Mg2+, the larger subunit of ribosome now
combines with 40S- mRNA- tRNAMet complex to form intact
ribosome.
 It requires initiation factor IF1 in prokaryotes and factors eIFl, eIF4 in
eukaryotes.
 Coming together of the two subunits of ribosomes is called
association.
 The intact ribosome encloses the mRNA-tRNA complex present at
the P-site but keeps the А-site exposed.
4. Elongation (Polypeptide Chain Formation)
(i) Binding of AA-tRNA at site-A
 An aminoacyl tRNA complex reaches the А-site and attaches to
mRNA codon next to initiation codon with the help of its anticodon.
 The step requires GTP and an elongation factor (eEFl in eukaryotes
and EF-Tu as well EF-Ts in prokaryotes).
 It has been found out that in Escherichia coli the most abundant
protein is elongation factor (EF-Tu).
(ii) Formation of peptide bond
 A peptide bond (-CO-NH-) is established between the carboxyl group
(-COOH) of amino acid attached to tRNA at P-site and amino group
(-NH2) of amino acid attached to tRNA at А-site.
 The reaction is catalyzed by the enzyme peptidyl transferase which
is an RNA-enzyme.
(iii) Movement of peptidyl tRNA from A-site to P-site
 The connection between tRNA and the amino acid at the P-site
breaks.
 The free tRNA of the P-site slips to E-site and from there to the
outside of ribosome with the help of G-factor.
 The А-site carries peptidyl tRNA complex.
 Soon after the establishment of first peptide linkage and slipping of
the freed tRNA of P-site, the ribosome or mRNA shifts slightly. The
process is called translocation. It requires a factor called
translocase (EF-G in prokaryotes and eEF2 in eukaryotes) and
energy from GTP.
 As a result of translocation, the А-site codon alongwith peptidyl-tRNA
complex reaches the P-site.
 A new codon is exposed at the А-site. It attracts a new aminoacyl
tRNA complex.
 The process of bond formation and translocation is repeated.
 One by one all the codons of mRNA are exposed at the А-site and
get decoded through incorporation of amino acids in the peptide
chain.
 The peptide chain elongates.
 The elongated peptide chain or polypeptide lies in the groove of the
larger subunit of ribosome to protect itself from cellular enzymes.
 A lot of energy is consumed in protein synthesis.
  For every single amino acid incorporated in peptide chain one ATP
and two GTP molecules are used.
 As one ribosome moves along the length of mRNA, the initiation
point on mRNA becomes free. It can form an initiation complex with
smaller subunit of another ribosome. In this way, a number of
ribosomes get attached to a single mRNA molecule. This complex is
known as polyribosome or polysome.
5. Termination
 Polypeptide synthesis is terminated when a nonsense codon of
mRNA reaches the А-site.
 There are three nonsense codons-UAA, UAG and UGA.
 These codons are not recognized by any of the tRNAs.
 Therefore, no more aminoacyl tRNA reaches the А-site.
 The P-site tRNA is hydrolyzed and the completed polypeptide is
released in the presence of GTP-dependent release factor.
 Release factors RF1 and RF2 are present in prokaryotes and eRFl in
eukaryotes.
  In prokaryotes RF1 is specific for UAG and UAA. RF2 is specific for
UAA and UGA.
 Ribosome moves over the nonsense codon and slips off the mRNA
chain.
 The two subunits of ribosome separate or undergo dissociation in the
presence of dissociation factor (DF).

Inhibitors of Protein Synthesis

Antibiotic Effect
Streptomycin Inhibits initiation of translation
 and causes misreading
Tetracycline Inhibits binding of aminoacyl-
tRNA to ribosome
Chloramphenico Inhibits peptidyl transferase and
l so formation of peptide bonds
Erythromycin Blocks formation of 70S ribosome
Neomycin Inhibits interaction of tRNA with
mRNA
Puromycin Binds to c-terminus of growing
peptide chain and causes
premature termination of protein
polypeptide chain in both
prokaryotes and eukaryotes