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Vipin Shankar

70s initiation complex 30s initiation complex
 Once the ribosome is assembled with the
charged tRNA in the P site, polypeptide
synthesis can begin.
 3 key events
 The correct amino acyl tRNA is loaded in the A site.
 A peptide bond is formed between the amino acyl
tRNA in the A site and peptide chain of the peptidyl
tRNA in the P site.
 Translocation of the resulting peptidyl tRNA from A
site to P site.
 As with the original positioning of the mRNA,
the shift must occur precisely to maintain the
correct reading frame.
 Two auxiliary proteins, Elongation factors,
control these events.
 Both elongation factors are GTP binding
Delivery of amino acyl tRNA to A site
 Amino acyl tRNA is ‘escorted’ to the ribosome by
the elongation factor Ef-Tu.
 Ef-Tu binds to the 3’ end of the tRNA masking
the coupled amino acid- this prevents formation
of peptide bonds outside the translation
 Ef-Tu binds and hydrolyses GTP; the type of
Guanine nucleotide bound to it governs its
 Only [Ef-Tu/GTP] can bind to amino acyl tRNA.
Delivery of amino acyl tRNA to A site…

 Hydrolysis of Ef-Tu bound GTP is triggered by
the factor binding center.
 Ef-Tu comes in contact with the factor binding
site only after the correct codon-anti codon
match is made.
 When Ef-Tu hydrolyses its bound GTP, any
associated amino acyl tRNA is released.
Selection of correct amino acyl tRNA
 The error rate of translation is 10-3 to 10-4.
 The ultimate basis for selection is the base
pairing between the charged tRNA and the
codon at the A site.
 But this alone is not responsible for the high
rate of accuracy.
Selection of correct amino acyl tRNA..
 Mechanism 1
 2 adjacent Adenine residues are present in the 16s
rRNA of the small subunit.
 These bases form a tight interaction with the minor
groove of each correct base pair formed between the
anti codon and the first 2 bases of the codon.
 Non-Watson base pairing form a minor groove which
is not recognized by the ‘A’ pair, and dissociation of
the aa-tRNA takes place.
 Correctly bound aa-tRNA has a decreased
dissociation rate.
Selection of correct amino acyl tRNA..
 Mechanism 2
 The GTPase activity of Ef-Tu depends on codon- anti
codon base pairing.
 Even a single mismatch leads to a dramatic reduction
in GTPase activity.
 This is an example of kinetic selectivity.
Selection of correct amino acyl tRNA..
 Mechanism 3
 Occurs after release of Ef-Tu.
 When the charged tRNA is first introduced its 3’ end is
distant from the site of peptide bond formation.
 To participate in the peptidyl transferase reaction, the
tRNA must rotate into the peptidyl transferase
reaction center of the large subunit – Accommodation.
 Incorrectly paired tRNAs cannot bear the strain during
accommodation and hence dissociates.
 Referred to as Proof reading.
Formation of the peptide bond
 This reaction is catalyzed by the 23s rRNA of
the large subunit.
 The exact mechanism is not yet determined.
 It is proposed that the 23s rRNA base pairs
between the CCA ends of the tRNAs at A site
and P site.
 This positions the α -amino group of the aa-
tRNA to attack the COO- group of the growing
polypeptide on the p-tRNA.
 These interactions also stabilize the aa-tRNA
after accommodation.
Peptide bond
 Once the peptidyl transferase reaction has
occurred, the tRNA in the P site is deacetylated
and the growing peptide chain is linked to the
tRNA in the A site.
 For a new round of peptide chain elongation,
the P site tRNA must move to the E site and the
A site tRNA to the P site.
 At the same time the mRNA must move by 3
nucleotides to expose the next codon.
 These movements are coordinated within the
ribosome and are collectively referred to as
 Once the growing peptide chain has been
transferred to the A site tRNA, the 3’ end of this
tRNA move to the P site in the large subunit,
while its anti codon end remains in the A site of
the small subunit.
 Similarly, the now deacetylated P site tRNA is
located in the E site of the large subunit and the
P site of the small subunit.
 Thus translocation in the large subunit
precedes that in the small subunit and the
tRNAs are said to be in ‘hybrid state’.
 Completion of translocation requires elongation
factor Ef-G.
 In the hybrid state the tRNA in the A site uncovers
the Ef-G-GTP binding site at the A site of the large
 When Ef-G-GTP binds, it comes in contact with the
factor binding center of the large subunit.
 This triggers the hydrolysis of GTP by Ef-G.
 Ef-G-GDP has a different conformation, which helps
it to reach the A site of the small subunit and trigger
the translocation of the small subunit.
 The movement is initiated by the interaction of
the Ef-G-GDP with the decoding center of the
small subunit.
 This causes a displacement of the A site tRNA
into the P site, with a related movement of the P
site tRNA to the E site.
 During the movement the mRNA is shifted by 3
base pairs.
 Essentially the mRNA is pulled along with the
moving A site tRNA.
GTP exchange
 Both EF-Tu- GDP and EF-G-GDP should be
converted to Ef-Tu-GTP and Ef-G-GTP, for
continuing elongation.
 EF-G has a lower affinity to GDP, and GDP is
released rapidly after hydrolysis.
 Ef-Tu requires a second protein EF-Ts (GTP
exchange factor).
Elongation in eukaryotes
 There is no difference between prokaryotic and
eukaryotic translation elongation.
 The factors are named differently
 Ef-Tu is called eEF1
 EF-G is called eEF2
 Release factors terminate translation in response to stop
 2 classes of release factors
 Class 1

2 types in prokaryotes, RF1 & RF2.

RF1 recognizes UAG & UAA, RF2 recognizes UGA &

Eukaryotes have only eRF1 – recognizes all 3 stop
 Class 2

RF3 in pk & eRF3 in ek.
 Regulated by GTP.
 Stimulate release of the class 1 factors.
 A region of the release factors called – peptide
anti codon composed of 3 amino acids recognize
the stop codon.
 All factors share a conserved glycine-glycine-
glutamine (GGQ) motif, essential for the release
of the polypeptide chain.
Release of class 1 releasing factors
 Accomplished by RF3.
 RF3 has high affinity to GDP, exist as RF3-GDP in free
 RF3-GDP binds to Class 1 RFs.
 Release of the peptide chain initiates conformational
change and GDP is exchanged for GTP, forming RF3-GTP.
 This causes a high-affinity interaction within the
ribosome causing the release of the class 1 RF.
 Release of RF brings about a conformational change and
RF3-GTP comes in contact with the factor binding site.
 GTP is hydrolyzed and RF3-GDP is released.
Ribosome Recycling.
 After the release of the polypeptide chain & the
release factor, the ribosome is still bound to the
mRNA and is left with 2 deacetylated tRNAs (in
the P site and E sites).
 To participate in new round of peptide
synthesis, the tRNAs and the mRNA must be
removed and the ribosome must dissociate into
the large & small subunits – ribosome recycling.
Ribosome recycling…
 Ribosome recycling factor (RRF) cooperates with
Ef-G and IF3 to recycle ribosome.
 RRF binds to the empty A site and mimics a tRNA.
 RRF also recruits EF-G to ribosome and, in events
that mimic elongation, the tRNAs at P site and E
site are released.
 Once the tRNAs are removed, RRF and EF-G are
released along with the mRNA.
 IF3 now binds to the small subunit and the
ribosome dissociate into the large and small
Post translational processing
 Final stage of protein synthesis.
 The nascent poly peptide chain is folded and
processed into its biologically active form.
 During or after its synthesis, the polypeptide
progressively assumes its native conformation
by forming hydrogen bonds, van der Waals’
linkages, hydrophobic interactions, hydrophilic
interactions etc.
 The linear or one dimensional genetic message
on the mRNA is converted to the three
dimensional protein.
Post translational processing..
 Amino terminal & carboxyl terminal
 Loss of signal sequences.
 Modifications of individual amino acids.
 Attachment of carbohydrate side chains.
 Addition of isoprenyl groups.
 Addition of prosthetic groups.
 Proteolytic processing.
 Formation of di-sulfide linkages.
Amino & Carboxyl terminal modifications

 The first aa inserted during translation is fmet
(in pk) & met (in ek).
 However, the formyl group, the amino terminal
met residue, and often additional amino
terminal residues are removed.
 Enzyme deformylase helps in the removal of
formyl group.
 Sometimes residues from the carboxyl terminal
are also removed.
Loss of signal sequences.
 15 – 20 residues at the amino terminal play a
role in directing the protein to the target – these
residues are called the signal sequence.
 Such signal sequences are ultimately removed
by specific peptidases.
Modifications of individual amino acids

 The OH- groups of certain Ser, Thr and Tyr of
some proteins are enzymatically
 Many proteins contain monomethyl and
dimethyl lysine.
 Calmodulin contains a trimethyl lysine at a
specific location.
 In some proteins the COO- of Glu undergo
 Attachment of carbohydrate side chains.
 Glycosylation plays an indispensable role in the
sorting and distribution of proteins.
 Polysaccharides linked to the amide nitrogen of
Asn confers stability on some glyco-proteins.
 Glycosylation plays an important role in cell
-cell adhesion.
 In some glycoproteins, the
carbohydrate side chain
is attached enzymatically
to Asn residues (N-linked
 In others to Ser or Thr
residues (O-linked
Addition of isoprenyl groups
 A thio-ether bond is formed between the isoprenyl
group and a Cys residue of the protein.
 The isoprenyl groups are derived from pyro-
phosphorylated intermediates of the cholesterol
biosynthetic pathway such as farnesyl
 Proteins modified in this way include the Ras
proteins, products of the ras oncogenes and proto-
oncogenes, and G proteins, and lamins, proteins
found in the nuclear matrix.
Addition of isoprenyl groups..
 The isoprenyl group helps to anchor the protein in a
 The transforming (carcinogenic) activity of the ras
oncogene is lost when isoprenylation of the Ras protein
is blocked, a finding that has stimulated interest in
identifying inhibitors of this posttranslational
modification pathway for use in cancer chemotherapy.
Addition of prosthetic groups
 Many prokaryotic and eukaryotic proteins
require for their activity covalently bound
prosthetic groups.
 Two examples are the biotin molecule of acetyl-
CoA carboxylase and the heme group of
hemoglobin or cytochrome c.
Proteolytic processing
 Many proteins are initially synthesized as large,
inactive precursor polypeptides that are
proteolytically trimmed to form their smaller,
active forms.
 Examples include pro-insulin, some viral
proteins, and proteases such as
chymotrypsinogen and trypsinogen
Formation of disulfide bridges
 After folding into their native conformations, some
proteins form intra-chain or inter-chain disulfide
bridges between Cys residues.
 In eukaryotes, disulfide bonds are common in
proteins to be exported from cells.
 The cross-links formed in this way help to protect
the native conformation of the protein molecule from
denaturation in the extracellular environment,
which can differ greatly from intracellular conditions
and is generally oxidizing.
Inhibition of translation
 Protein synthesis is a central function in cellular
physiology and is the primary target of many
naturally occurring antibiotics and toxins.
 Natural selection has favoured the evolution of
compounds that exploit minor differences in order to
affect bacterial systems selectively, such that these
biochemical weapons are synthesized by some
microorganisms and are extremely toxic to others.
 Because nearly every step in protein synthesis can
be specifically inhibited by one antibiotic or another,
antibiotics have become valuable tools in the study
of protein biosynthesis.
Inhibitors of translation
 Puromycin
 Tetracycline
 Chloramphenicol
 Cyclohexamide
 Streptomycin
Inhibitors of translation…
 Puromycin
 Made by the mold Streptomyces alboniger.
 Its structure is very similar to the 3’ end of an amino
acyl-tRNA, enabling it to bind to the ribosomal A site
and participate in peptide bond formation, producing
 However, because puromycin resembles only the 3
end of the tRNA, it does not engage in translocation
and dissociates from the ribosome shortly after it is
linked to the carboxyl terminus of the peptide.
 This prematurely terminates polypeptide synthesis.
Peptidyl transferase
Inhibitors of translation…
 Streptomycin
 A basic trisaccharide, causes

misreading of the genetic
code (in bacteria) at relatively
low concentrations and
inhibits initiation at higher
Antibiotic/Tox Target Molecular Consequences
in cells Target
Tetracycline Pk A site of 30s Inhibits amino-acyl tRNA binding
subunit to the A site
Hygromycin B Pk & Near the A site Prevents translocation of A site
Ek of 30s subunit tRNA to P site
Paromycin Pk Adjacent to the A Increases error rate of translation
site codon-anti by decreasing selectivity of
codon codon-anti codon pairing.
interaction site
Chloramphenic Pk inPeptidyl
the 30s Blocks correct positioning of the A
ol subunit
transferase site amino-acyl tRNA for peptidyl
center of 50s transfer reaction.
Puromycin Pk & subunit
Peptidyl transfer Chain terminator- mimics the 3’
Ek center of large end of amino acyl tRNA in A site
ribosomal and acts as acceptor for the
Erythromycin Pk subunit
Peptide exit nascent polypeptide
Blocks exit chain.
of the growing
tunnel of 50s polypeptide chain from the
ribosome; arrests translation.
Fuisidic acid Pk Ef-G Prevents release of Ef-G-GDP
from the ribosome.
Antibiotic/Tox Target Molecular Consequences
in cells Target
Thiostrepton Pk Factor binding Interferes with the association of
center of 50s IF2 and EF-G with the factor
Kirromycin Pk & subunit
Ef-Tu binding
Prevents center.
Ef-Tu release.
Ricin and α- Pk & Chemically Prevents the activation of
Sarcin (Protein Ek modifies the RNA translation factor GTPase.
toxins) in the factor
binding center of
Diptheria toxin Ek theChemically
large subunit Inhibits EF-Tu function.
modifies Ef-Tu
Cyclohexamide Ek Peptidyl Inhibits peptidyl transferase
transferase activity.
center of the 60s