TRANSLATION

Gopukrishnan R
CADD
Department of Computational Biology and
Bioinformatics, UoK
TRANSLATION
 Translation means that genetic information
copied into RNA with transcription is
converted to a protein or polypeptide chain. In
other words, it is the expression of genetic
information in the form of protein molecules.

The process of translation involves several key steps:

2
Translation Initiation

3
Key component required for translation initiation

1. mRNA 5’ 3’

2. 30s & 50S ribosomal subunits (prokaryotes)

Large
subunit

60S & 40S ribosomal subunits (eukaryotes) Small

subunit

3. tRNA (amino acid binding site and anticodon)

- A specific charged tRNA (initiator tRNA), f-Met-tRNA

4. Guanosine triphosphate (GTP) required for the assembly

5. A set of soluble protein factors — initiation factors (IF, in eukaryotes –eIF)

G-protein family members that use the energy released upon

hydrolysis of bound GTP. When they are bound to GTP they are consider to be
active.

4
1. mRNA

5’ 3’

The AUG initiator codon is

like the launchpad for
protein synthesis, signalling
the start of translation. It's
the first step in building a
protein.
5
 2. Ribosomal subunit
Eukaryotes Ribosome Prokaryotes

28S rRNA 23S rRNA

60S 5.8S rRNA Large 5S rRNA 50S
Subunit 5S rRNA subunit 34 proteins Subunit
80S 50 proteins
70S

Small 16S rRNA

18S rRNA 30S
40S subunit 21 proteins
35 proteins Subunit
Subunit

The relative sizes of ribosomal subunits are given in terms of their sedimentation
coefficient or S(Svedberg) valves, a non-linear function that measures time-based
on speed.
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Important binding sites on ribosomes

Peptidyl site (P or parking site):

Occupied by the peptidyl-tRNA,
the tRNA carrying the growing
polypeptide chain

Exit site (E): Acceptor site (A):

The site where the empty Attachment site for an
tRNA (unloaded or incoming aminoacyl- tRNAs
deacylated tRNA)occupies
prior to exiting the ribosome
E P A

5’ 3’
mRNA

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mRNA recognition and alignment
To distinguish initiation codon and other methionine, prokaryotes contain a specific
sequence, Shine Dalgarno sequence (AGGAGGU), which is located about 5-10 nucleotides
before the initiation codon. Close to the 3’ of 16S rRNA of the 30S ribosomal subunit, there is
a nucleotide sequence complementary to the Shine-Dalgarno sequence.

Prokaryotes
Initiation
codon
Shine-Dalgarno
(SD) sequence
mRNA
5’ 3’

3’
5’

16S rRNA

30S ribosomal subunit in 8

mRNA recognition and alignment
Eukaryotic mRNAs first interact with the ribosome with the 5’ cap structure; once the ribosome
recognises the cap structure, it moves to the 5’-AUG-3’ initiation codon. During movement, with the help
of small subunit initiation factors, the initiation codon in mRNA (AUG codon) with the base match with the
anticodon in initiator tRNA is found. There are two other inducing translations of eukaryotic mRNA; one is
in some mRNAs, there is a purine three bases upstream of the initiation codon and a guanine downstairs
(5’-RCCAUGG-3’) (Kozak sequence).

Eukaryotes
eIF-4
5’ cap
Kozak sequence
5’ 7 𝐺 AAAAAAAAA 3’
:-7-Methylguanosine marks
𝑚
the beginning of mRNA
molecules as the cap
structure.
Poly A tail
40S ribosomal subunit in Eukaryotes The poly-A tail is made up
of a series of adenine
nucleotides (A) added to
the 3' end of the mRNA
molecule during
transcription. 9
30S ribosomal subunit in prokaryotes

Kozak sequence

40S ribosomal subunit in Eukaryotes

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3. Initiator tRNA()
• , is a tRNA which reads AUG sequence on mRNA that signals start site.
• Only reads the start codon (AUG), not the internal codon (AUG GGU UCG AUG UUU).
• Essential for translation initiation but inappropriate for peptide chain elongation.
• Eukaryotic tRNA carries non- formylated methionine () , where as prokaryotes tRNA initially
have formylated methionine.

Generation of the initiator N-formyl-methionyl-tRNA()

N-Formyl-
methionine(fMet)

Methionine
Methionyl-formyl transferase

10 THF
𝑁 − 𝑓𝑜𝑟𝑚𝑦𝑙 − 𝑇𝐻𝐹
tetrahydrofolate

𝑡𝑅𝑁𝐴
𝑀𝑒𝑡
Initiator 11
4. Soluble protein factors & GTP

Prokaryotes Eukaryotes Function

 Hydrolysis of GTP to GDP+Pi provide

Initiation factors(IFs)
IF-2-GTP eIF2-GTP the energy for the assembly of
bound to initiator tRNA
initiation complex
• Facilitates binding of small subunit to
IFs bound to small eIF1, eIF1A initiator tRNA
IF-1, IF-3
ribosomal subunit eIF3, eIF5 • Facilitate base pairing between
anticodon & codon

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Formation of initiation complex in prokaryotes

Small subunit Initiator

bound to IF tRNA
mRNA

IFs

Large
subunit
IF-GDP + Pi

IFs

Complete initiation complex 13

Translation Elongation

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Key component required for peptide chain elongation

1. A mRNA : 70s(Pro) / 80s(Euk) ribosome : initiation

complex
2. Aminoacyl-tRNAs

 tRNAs carrying amino acids.

 Amino acids- “activated”

 tRNA – “charged”(tRNA with amino acid)

What is activated amino acid and charged
tRNA in translation?
3. A set of soluble proteins- “elongation factors (EF)” The process of attaching an
amino acid to its corresponding transfer
RNA (tRNA) is called amino acid activation,
4. Guanosine triphosphate (GTP) also known as tRNA charging or
aminoacylation.

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Activation of amino acid by aminoacyl-tRNA synthetase
Why is it necessary to activate amino acids
PPi during translation?
To ensures that the correctP amino P
acids are recognised and
that there is enough energy to form peptide bonds.
P P P
O P

Aminoacyl-tRNA
Amino acid synthetase
ATP aminoacyl adenylate intermediate
(AMP-amino acid)
Aminoacyl-tRNA
synthetase

AMP
tRNA

Aminoacyl-tRNA
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 A

Aminoacyl-tRNA can occur in 2 ways

1. Amino acid 3’OH of ribose on Adenosine

nucleotide Class II Aminoacyl-
tRNA
Aminoacyl-tRNA Synthetase (20 synthetase
enzymes, 1 for each amino acid),
Aminoacyl-tRNA
1. Activates the amino acids.
2. Amino
2. Differentiates acid amino acids(20) & tRNAs(50
between 2’OH of ribose
Euk. on40
& 30- Adenosine
Pro.) to synthesize
nucleotide
aminoacyl-tRNA Class I Aminoacyl-
tRNA synthetase (2’-aminoacyl ester)

Transesterification

3’OH of ribose on Adenosine nucleotide

Only the 3’- esters are substrates for protein synthesis 17
Codon – anticodon pairing tRNA
Anticodon

3 2 1

3’ U U C 5’

1. Codon and anticodon pairing is “antiparallel”

2. tRNA recognize more than one codon for a given amino acid. This means
that amino acids can be coded for by more than one codon. For example, 5’ A
1
A
2
G
3
3’

the tRNA for phenylalanine can pair with either the 5'-UUC-3' or 5'-
UUU-3' mRNA codons, both of which specify phenylalanine. codon
mRNA

tRNA
Anticodon
3. Codon – anticodon pairing is “Wobble”. Wobble pairing is another factor
that allows tRNA to recognize multiple codons for the same amino acid. 3 2 1

The last base in a codon, known as the wobble base, is responsible for 3’ A G G 5’

degeneracy.

5’ U C C/U 3’
The Wobble Hypothesis describes how one 1 2 3

amino acid can be coded for by several

codons. The third nucleotide in a codon is codon
mRNA
referred to as the wobble position.
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Wobble hypothesis
Francis Crick first proposed the Wobble Hypothesis in 1966

4 Relationships

1. The first two bases in the mRNA molecule will be responsible for strong
Watson and Crick pairing.
A T G
“Strong
binding ”
GC A
GC G
GC C

Alanine GC U
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 Wobble hypothesis
2. One mRNA molecule’s codon is in line with the anticodon of that tRNA molecule.
The first base of the anticodon and the third base of the codon are responsible
for the number of codons recognized by the tRNA molecule.

tRNA

3’ 3 2 1 5’
#1 base (Anticodon) responsible for the
number of codons
#3 base (Codon) recognized by the
tRNA molecule.
5’ 1 2 3
3’
mRNA

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1. One codon:-

Anticodon XX- C XX- A

Codon XX- G XX- U

2.Two codons:-

Anticodon XX- U XX- G Uracil can also form a non-standard

hydrogen bond with guanine. This
Codon XX- A/G XX- C/U pairing is less stable than the
standard pairing but still sufficient
to allow proper translation of the
3. Three codons:- Wobble Hypothesis genetic code.

Anticodon XX- I Inosine

Codon XX- A,U,C

hypoxanthine

Inosine Monophosphate 21
 Wobble hypothesis

3. When one amino acid has several codons, the difference in

either the first or two bases will require a separate tRNA
molecule.
4. There are a minimum of 32 tRNA molecules for 61 codons,
and three codons, as we know, are stop codons.

Why did the tRNA molecule feel

overwhelmed? 1.
Because it had to handle 61 codons Initi
ator
with just 32 anticodons comp
lex

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 Initiator
tRNA
2. Loading of
(In Eukaryotes –eEF1A) aminoacyl-tRNA
EF-Tu-GDP + Pi on A site

1. Initiator complex

Unloaded or
uncharged tRNA
Elongation 3. Peptide bond
formation
Peptidyl transferase
(by 23s rRNA ribozyme)

EF-TU-GTP

EF-G-GDP + Pi EF-G-GTP Peptide bond

4. Translocation
5‘ 3‘

EF-Tu-GDP + Pi (In Euk— eEF2, eukaryotic

translocation factor) 23
 Peptidyl translocation

GT
transferase

GD
P
+
Pi
Elongation

E P A

After several steps

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Translation Termination

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 Release factor(RF):
RF1(UAG,UAA)
RF2(UGA)
(In Euk. eRF)

1. Termination

Termination Codon
(UAG,UAA,UGA)
Termination
2. Complex
protein
dissociation

RF3-GTP

RF

RF3-GDP + Pi
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