TRANSLATION

Dr Mukhtiar Baig
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 Learning Objectives
• Understand the general characteristics of the
genetic code
• Know the start and stop codons
• Understand the wobble hypothesis and its
importance
• Understand the structure of the ribosome
• Understand the role of mRNA, tRNA and rRNA in
protein synthesis
• Understand the steps of translation
• Understand the importance of posttranslational
modifications

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 Books recommended for
this topic
• Lehningertext book of biochemistry
(excellent)
• Harper’s biochemistry
(good)
• Lippincottsbiochemistry
(good)

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 The genetic code
• Dictionary that identifies the
correspondence between a sequence
of nucleotide bases and a sequence of
amino acids.

• Each individual word in the code is

composed of three nucleotide bases.

• These genetic words are called

codons.

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• Codons are presented in the
messenger RNA (mRNA) language of
adenine (A), guanine (G), cytosine
(C), and uracil (U).

• Nucleotide sequences always from

the 5′-end to the 3′-end.

• The four nucleotide bases produce

the three-base codons.

• 64 different combinations of bases.

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 How to translate a
•
codon:
This table (or “dictionary”) can be
used to translate any codon sequence
and, to determine which amino acids
are coded for by an mRNA sequence.

• Eg. The codon 5′-AUG-3′ codes for

methionine.

• Sixty-one of the 64 codons code for

the twenty common amino acids

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 Codons With Special
Roles
Start AUG
Codon:
Stop
Codons:
UAA UAG UGA

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 Characteristics of the
genetic code
1. Universality:
3. Degeneracy:
3. Specificity:
4. Nonoverlapping and nopunctuation:

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 Wobble hypothesis

• “The third base of the tRNA anticodon does

not have to pair with a complementary codon
(as do the first two) but can form base pairs
with any of several related codons.”

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 Wobble hypothesis

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 Messenger RNA (mRNA)
start
codon

mRNA A U G G G C U C C A U C G G C G C A U A A

codon 1codon 2codon 3codon 4codon 5codon 6codon 7

protein met gly ser ile gly ala stop
codon
Primary structure of a protein

aa1 aa2 aa3 aa4 aa5 aa6

peptide bonds
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 tRNA

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 Ribosomal Subunits

Complete
L functional
Large
subunit Small ribosome
subunit

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Ribosomal RNA

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 Protein Synthesis
Takes place in five stages
• Activation of precursors
• Initiation
• Elongation
• Termination
• Folding and Posttranslational
Processing

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Components Required for the Five Major
Stages of Protein Synthesis in E. coli
1. Activation of 2. Initiation mRNA
amino acids • N-
• 20 amino acids Formylmethionyl-
• 20 aminoacyl- tRNAfmet
tRNA synthetases • Initiation codon in
• 32 or more tRNAs mRNA (AUG)
• ATP • 30S ribosomal
• Mg+2 subunit
• 50S ribosomal
subunit

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3. Elongation 4. Termination
• Functional 70S and release
ribosome • Termination
(initiation codon in mRNA
complex) • Release factors
• Aminoacyl- (RF-1, RF-2, RF-
tRNAs 3)
specified by
codons

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 5. Folding and
• posttranslational
Specific enzymes, cofactors, and
other components for removal of
initiating residues and signal
sequences,
• Additional proteolytic processing,
• Modification of terminal residues, and
• Attachment of phosphate, methyl,
carboxyl, carbohydrate, or prosthetic
groups

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 Stage 1: Aminoacyl-tRNA
synthetases attach the correct
• amino
Aminoacids
acidto
+their
tRNA tRNA
+ ATP

Mg2+ Aminoacyl- tRNA

synthetase

Aminoacyl-tRNA + AMP + PPi

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 M
• Amino acid + tRNA + ATP
g2+
aminoacyl-
tRNA + AMP + 2Pi
ΔG0
≈ -29 kJ/mol

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 Amino acid
R O uncharged tRNA
H2N-C-C-OH

=
- -
3’
ATP H
adenylated (activated)
amino acid
RO
=
- -

H2N-C-C-O-P-O-ribose-adenine
PPi
H

AMP RO
H2N-C-C-O

=
- -
Amino acid activation H
and tRNA charging aminoacyl
(charged)
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tRNA 1
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 Aminoacyl-tRNA
3’ end of tRNA synthetases

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 3’ end of
tRNA

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Stage 2: A Specific Amino Acid
Initiates Protein Synthesis

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Formation of the initiation
complex takes place in
• In step 1 the 30S ribosomal subunit
binds two initiation factors, IF-1 and
IF-3, then mRNA binds to the 30S
subunit.
• In step 2 GTP-bound IF-2 and the
initiating fMet-tRNAfMet are joined to
the complex
• In step 3 this large complex combines
with the 50S
ribosomal subunit.
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 Protein factors required for initiation of
translation in bacterial and eukaryotic cells
Bacterial

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EUKAROYTIC

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 Stage 3: Elongation Stage

It completes in three steps

• 1. Binding of an Incoming
Aminoacyl-tRNA
• 2. Formation of peptide bonds
• 3. Translocation

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Binding
of
incomin
g
aminoac

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 Peptide
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 Peptide bond
formation

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Translocation

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 Incoming
aminoacyl-
tRNA3

Direction of
ribosome movementBUMDC
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 Stage 4: Termination of
Polypeptide Synthesis
Release factor
binds

Polypeptidyl- tRNA
link hydrolyzed
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 Polypeptidyl-tRNA
link hydrolyzed

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 Components
dissociate

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 Stage 5: Folding and
• BeforePosttranslational
or after folding, the new
polypeptide may undergo enzymatic
processing, including
• Removal of one or more amino acids;
• Addition of acetyl, phosphoryl,
methyl, carboxyl, or other groups to
certain amino acid residues;
• Proteolytic cleavage; and/or
• Attachment of oligosaccharides or
prosthetic groups.

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 Phosphorylation
dephosphorylation

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Carboxyation

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 Loss of Signal Sequences
N C-peptide
H + C-peptide
Signal 3
sequence N
H3+ -S
-S S-
-SH HS-- SH N
B-chain -SH -S H3+ -S
-S S- -S S-
-SH HS-
COO- -S
COO- -S S-
A-chain
C
OO-
PROINSULIN INSULIN
PREPROINSULIN

Insulin and its precursor forms

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 Proteolytic
cleavage

Enterokinase

Trypsinogen Trypsin

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 Phosphorylation

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 Hydroxylation

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 Biotinylated enzyme

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 Farnesylated
protein

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 Glycosylation

N-acety-glucosamine
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 Formation of Disulfide
Cross-Links

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Conversion of proinsulin
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 Inhibitors of Protein
Synthesis
Acting only on bacteria
• Tetracycline: blocks binding of
aminoacyl tRNA to A site of
ribosomes.
• Streptomycine: prevents the
transition from translation initiation
to chain elongation also causes
miscoding
• Chloramphenicol: bolcks the peptidyl
tranferase reaction on ribosomes

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• Erythromycin: binds in the exit
channel of ribosomes thereby inhibits
elongation of the peptide chain
• Rifamycin: blocks initation of RNA
chains by binding to RNA polymerase
(prevent RNA synthesis).

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 Acting on bacteria and
eukaryotes
• Puromycin: causes the premature
release of nascent polypeptide chains
by its addition to the growing chain
end
• Actinomycin D: binds to DNA and
blocks the movement of RNA
polymerase (prevent RNA synthesis).

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 Acting on eukaryotes but
not bacteria
• Cycloheximide: blocks the
translocation reaction on ribosomes.
• Anisomycin: bolcks the peptidyl
transferase reaction on ribosomes.
• α Amanitin: blocks mRNA synthesis by
binding preferentially to RNA
polymerase II.

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• Some of these antibiotics have
deleterious effects on human
mitochondria because of their
resemblance with the bacteria in
their sensitivity to inhibitors. .

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Tetracycli Chloramphen
ne Spectinom icol
ycin

Hygromyc
in B Erythromy
Streptom Streptogra cin
ycin min B
Small ribosomal Large ribosomal
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subunit subunit
 The Action of Antimicrobial

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Figure 20.4