Translation

TRANSLATION
Department of Biochemistry, KMC, DuwakotTuesday, December
23, 2014
Rajesh Chaudhary 1

“An understanding of protein synthesis, the
most complex biosynthetic process, has been
one of the greatest challenges in
biochemistry” – Lehninger
Translation
Tuesday, December 23, 2014
2
Rajesh Chaudhary

Translation / Protein synthesis
Tuesday, December 23, 2014Rajesh Chaudhary
3
 Translation: Language of nucleotide
sequence is translated into the language of
an “amino acid” sequence.
 Genetic code: …through which information
can be translated from nucleotide to amino
acid sequence.

The Genetic Code
4
 A “codon” is a triplet of nucleotides that codes for a specific
amino acids.
CODON
 Codon are presented in mRNA language of Adenine (A),
Guanine (G), Cytosine (C), and Uracil (U).
 Written in 5’ 3’ direction.
 Four nucleotide bases are used to produce 3 base codon –
thus, 64 total combinations.

How to translate a codon?
5
Termination, Stop or Non-sense codon and how frequently they are encountered?

Characteristics of the genetic code
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 Specific / Unambiguous
 Universal
 Degenerate / Redundant
 Nonoverlapping and commaless
ABCDEFGHIJKL is read as ABC/DEF/GHI/JKL

Reading frame in genetic code
7
What is an Open Reading Frame (ORF)?

Consequence of altering the
nucleotide sequence
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 Point mutation / single nucleotide base on mRNA
leads to the following three conclusions:
 1. Silent mutation
 2. Missense mutation
 3. Nonsense mutation
 4. Other mutations
 4.1. Trinucleotide repeat expansion
 4.2. Splice site mutation
 4.3. Frame-shift mutation

Role of tandem triplet repeats in mRNA causing
Huntington disease and other triplet expansion
diseases
9
Frame-shift Mutation

Wobble Hypothesis
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Wobble allows some tRNA to recognize more than one codon.
When several different codons specify one AA, the
difference between them usually lies at the third
base.
Example: Alanine is coded by GCU, GCC, GCA and
GCG.

Crick proposed four relationships
called “Wobble Hypothesis”11
 1. The first two bases of an mRNA codon always form strong
Watson-Crick base pairs with the corresponding bases of the
tRNA anticodon and confer most of the coding specificity.
 2. The first base of the anticodon determines the number of
codons recognized by the tRNA.
 3. When an AA is specified by several different codons, the
codons that differ in either of the first two bases require different
tRNAs.
 4. A minimum of 32 tRNAs are required to translate all 61
codons (31 to encode the AA and 1 for initiation).

Wobble Hypothesis
Rajesh Chaudhary
12
How the Wobble Base of the Anticodon Determines the Number of Codons a tRNA
Can Recognize

Components required for
“translation”
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 1. Amino acids
 2. Transfer RNA
 Amino acid attachment site
 Anticodon
 3. Aminoacyl–tRNA synthetase
 4. Messenger RNA
 5. Functionally competent ribosomes
 6. Protein factors
 7. ATP and GTP are required as a
source of energy

2. Transfer RNA (tRNA)
14
 There are at least 50 species of tRNA in
humans, and 30-40 species in bacteria.
 One AA have more than one tRNA to
carry it.
 2.1. Amino acid attachment site
 2.2. Anticodon
When tRNA has covalently attached AA, it is said to
be charged or else uncharged !

3. Aminoacyl-tRNA synthetase
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 The family of this enzyme is
necessary for attachment of AA
to its corresponding tRNA.
 Catalyze two-step reaction
leading to attachment of AA.
 Synthetase have “proofreading”
or “editing” activity that can
remove AA from enzyme or
tRNA.

4. Messenger RNA (mRNA)
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 The specific mRNA required as a template for the
synthesis of the desired polypeptide chain must be
present.
 Interactions between proteins that bind the 5’-cap (elF-4
protein) and the 3’-tail (poly-A binding proteins) of
eukaryotic mRNA mediate circularization of the mRNA and
likely prevent the use of incompletely processed mRNA in
translation.

5. Functionally competent ribosome
17
Prokaryotic Vs Eukaryotic Ribosomes
What is the function of smaller and larger subunits of ribosomes?

5. Functionally competent Ribosomes
(contd…)
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 5.1. Ribosomal RNA
 5.2. Ribosomal proteins
 5.3. A, P and E sites on
Ribosome
 5.4. Cellular locations of
Ribosomes
1. A-site: binds incoming
Aminoacyl-tRNA
2. P-site: occupied by
Peptidyl transferase
3. E-site: occupied by Empty
tRNA

6. Protein factors
19
 Initiation, elongation and termination factors are
required for peptide synthesis.
 Some provide catalytic function while others work in
stabilization process of complex.
 NOTE: Numbers of factors are G-protein, thus
active when bound to GTP and inactive when
bound to GDP.

7. ATP and GTP are required as the
source of energy
20
 Cleavage of four high energy bonds is required for
addition of one AA to the growing polypeptide
chain – two from ATP and two from GTP.
 Additional ATP and GTP are required for initiation
in eukaryotes while additional GTP is required for
termination in both Prokaryotes and Eukaryotes.

Steps in Protein synthesis
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 mRNA is translated from its 5’ end to its 3’ end.
 Prokaryotic mRNA are called “polycistronic” because they
have more than one coding regions.
 Each coding regions have their own initiation and termination codons
and produces separate piece of polypeptide chains.
 Eukaryotic protein synthesis resembles that of prokaryotes in most
aspects, however, there are still some differences.
Initiation, Elongation and Termination

1. Initiation
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 In prokaryotes 3 initiation factors are known: IF-1, IF-2 and IF-
3.
 In eukaryotes, there are over 10 initiation factors: eIF…
 There are two mechanisms by which ribosome recognizes
nucleotide sequence (AUG) that initiates translation:
 1. Shine-Dalgarno Sequence.
 2. Initiation Codon

Complementary binding between prokaryotic mRNA
Shine-Dalgarno sequence and 16S rRNA
23

Initiation codon
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 Initiating “AUG” is recognized by
special initiator tRNA.
 Recognition is facilitated by IF-2
(bound to GTP) in Prokaryotes and
eIF2-GTP in Eukaryotes.
 The AA charged initiator tRNA enters
the ribosomal P site, and GTP is
hydrolysed to GDP.
NOTE: The initiator tRNA is the only tRNA recognized by eIF-
2 and the only tRNA to go directly to the P site.

Elongation
25
 The delivery of aminoacyl-tRNA whose
codon appears next on the mRNA template
in the ribosomal A site is facilitated in E. Coli
by elongation factors EF-Tu & EF-Ts and
require GTP hydrolysis.
 In Eukaryotes, comparable elongation factors
are: EF-1a & EF-1bg.
 Translocation: Shifting of ribosome three
nucleotide towards 3’-end of mRNA.
 In Prokaryotes it requires EF-G
 In Eukaryotes, it requires EF-2.
Because rRNA catalyze this reaction, it is known as “RIBOZYME”.

Termination
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 Termination of translation occurs when any one of the three
termination codons moves into the A-site.
 Codons are recognized in E. Coli by release factors:
 RF-1: recognizes termination codon UAA & UAG.
 RF-2: recognizes termination codon UGA & UAA.
 RF-3: (bound to GTP) causes release of RF-1 & RF-2 as GTP is
hydrolyzed.
 Eukaryotes have single release factor: eRF – which recognizes all
three codons.
Ricin (from Castor beans) is a very potent toxin that exerts its effects by removing
an adenine from 28S ribosomal RNA, thus, inhibiting eukaryotic ribosomes.

Polysomes & Protein targeting
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Regulation of Translation
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 Through covalent modification of eIF-2 –
phosphorylated eIF-2 is inactive.
 In both Prokaryotes and Eukaryotes regulation can
be achieved through proteins that bind to mRNA or
either inhibiting its use by blocking translation or
extending its use by protecting it from degradation.

Posttranslational modification of
polypeptide chains
29
 A. Trimming
 B. Covalent alterations
 1. Phosphorylation
 2. Glycosylation
 3. Hydroxylation
 4. Other covalent modification
 C. Protein degradation

References
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Translation

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