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FROM GENE TO PROTEIN

TRANSLATION

Protein synthesis is based on the


sequence of mRNA, which is made
up of nucleotides while proteins
are made up of amino
acids. There must be a specific
relationship between the
nucleotide sequence and amino
acid sequence. This relationship
is the so called genetic code
(Marshall
Nirenberg,1960s).

TRANSLATION
the whole process by which the nucleotide sequence of an mRNA is
used to order and to join the amino acids in polypeptide chain.
In eukaryotic cells, protein synthesis occurs in the cytoplasm, where
three types of RNA molecules come together to perform different
but cooperative functions

COMPONENTS OF TRANSLATION

1.
2.

mRNA
tRNA. Each AA has its own subset of tRNAs. each specific
tRNA molecule contains a three-nucleotide sequence, an
anticodon, that can base-pair with its complementary codon in
the mRNA

TRANSLATION
Component of
translation:
-mRNA
-tRNA
-Ribosom

3. rRNA associates with a set of protein to form ribosomes that


move along an mRNA molecule, catalyze the assembly of AA
into polypeptide chains. They also bind tRNA and various
accessory proteins necessary for protein synthesis.
- Ribosomes are composed of a large and small subunit, each of
which contains its own rRNA molecules.

tRNA

Primary and secondary structure of


tRNA

- a hairpin structure
- amino site at (3 end)
- anticodon site, that
bind with codon in
mRNA
There are 61 type tRNA
in cell

Amino Acid activation

Editing tRNA synthetase ensure


accuracy

AA is activated for protein synthesis by an amino acyl tRNA


synthetase

Anticodon
made up of three nucleotides correspond to the
three bases of the codon on the mRNA. Each tRNA
contains a specific anticodon triplet sequence that
can base-pair to one or more codons for an amino
acid.

There are 61 type tRNA in cell. what


about those having less than 61
tRNA?

ex, one codon for lysine is AAA; the anticodon of a lysine


tRNA might be UUU. Some anticodons can pair with more
than one codon due to a phenomenon known as wobble
base pairing.

Wooble theory

What about cell having < 61 types tRNAs ?


Wobble base
Frequently, the first nucleotide of the
anticodon is one of two not found on mRNA:
inosine and pseudouridine, which can hydrogen
bond to more than one base in the
corresponding codon position. In the genetic
code, it is common for a single AA to occupy all
four third-position possibilities;

Ribosome Structure

example, the amino acid glycine is coded for by the


codon sequences GGU, GGC, GGA, and GGG.

mRNA is associated with the 30S


subunit
Two tRNA binding sites (P and A
sites) are located in the cavity
formed by the 2 subunits.
The growing peptide chain
threads through a tunnel that
passes through the 40S (30S in
bacteria) subunit.

Ribosome structure

Translation Machinery in Prokaryotes (for


comparing with Eukaryotes)
Ribosomes:
-70S (L (50S) and S (30S) subunits)
-contain 23S (L), 16S (S), and 5S (L) rRNAs
-each subunit (L and S) contains ~30 proteins
Initiation factors: if1, if2, if3
Elongation factors: ef-Tu, ef-Ts, and G
Termination (release) factor(s): Rf1 and Rf2
Translation is initiated with fmet (N-formylated
methionine).

Identification of Initiator Codon in


Prokaryotes
Involves binding of initiator tRNA (N-formylmethionyl-tRNA) to
initiator codon (first AUG)
The 30S subunit scans the mRNA for a specific sequence (SD
Sequence) upstream of the start codon. 16S RNA is involved in
recognition of S-D sequence.

Mechanism of Protein Synthesis


three phases: initiation, elongation, termination
Initiation : binding of mRNA and initiator
aminoacyl-tRNA to small subunit, followed by
binding of large subunit
Elongation: synthesis of all peptide bonds - with
tRNAs bound to acceptor (A) and peptidyl (P) sites.
Termination occurs when "stop codon" reached

How is right AUG selected for translation in


Prokaryotes?
1.

Many mRNAs contain a sequence preceding the start


codon that base-pairs with the 3'-end of 16S rRNA
(Shine-Dalgarno sequence)

start
5'----GGAGG-------AUG-----3 mRNA
3'----CCUCC--------5'
16S rRNA
2.
3.
4.

Function: helps position mRNA in ribosome.


The AUG itself is also very important
There is a S-D independent mode of translation
initiation in E. coli
Translate internal ORFs of polycistronic mRNAs

Prokaryotic Translational Initiation


Formation of Initiation
complex (IC) involves IFs
IF-3 keeps ribosome subunits
apart
IF-2 identifies and binds
initiator tRNA. IF-2 must
bind GTP to bind tRNA.
IF-1, IF-2, and IF-3 bind to
30S subunit to form IC
Once 50S subunit binds IC,
GTP is hydrolyzed, initiator
tRNA enters P-site and IFs
dissociate

Elongation Factor Tu (EFTu) binds to aminoacyltRNA and delivers it to the


A site of the ribosome
When EF-Tu binds GTP a
conformational change
occurs allowing it to bind
to aminoacyl-tRNA.

Recycling of EF-Tu
After leaving the
ribosome EF-Tu-GDP
complex associates with
EF-Ts causing GDP to
disassociate.
When GTP bind to the
EF-Tu/EF-Ts complex,
EF-Ts disassociates and
EF-Tu can bind another
tRNA

Formation of Peptide
Bond
Once the peptide bond forms,
the mRNA shifts to move the
new peptidyl-tRNA to P-site
and moves the deaminacyltRNA from E-site
Binding of EF-GTP to
ribosome promotes the
translocation
Hydrolysis of EF-GTP to EFGDP is required to release EF
from ribosome and new cycle
of elongation could occur

Chain Elongation
Three step process:
1) Position correct aminoacyl-tRNA at acceptor site
2) Formation of peptide bond between peptidyl-tRNA
at P site with aminoacyl-tRNA at A site.
3) Shifting mRNA by one codon relative to ribosome.

EF-Tu-tRNA complex
enters the ribosome and
positions new tRNA at A
site.
If the anticodon matches
the codon, GTP is
hydrolyzed and EF-Tu
releases the tRNA and
then exits the ribosome.

Peptide Bond formation


P-Site
5' tRNA

H
O

H
OH

5' tRNA

5' tRNA

H
OH

OH

H
OH

5' tRNA

N
O

H
OH

C
C

CH H
CH H
H

H
BASE

CH H
NH3 +

More on elongation
Growing peptide chain extends
into the tunnel of 50S subunit.
Folding of the native protein
does not occur until the peptide
exits the tunnel
Folding is facilitated by
chaperones that are associated
with the ribosome
To ensure the correct tRNA
enters the A site, the 16S RNA is
involved in determning correct
codon/anticodon pairing at
positions 1 and 2 of the codon.

NH2

O
H

A-Site

NH2
N

CH H
NH3 +

O
H

P-Site

NH2

H+

A-Site

NH2
N

N
N

Peptide Chain Termination


Release factors" recognize the stop codon (UGA,
UAG, or UAA) at the A site
In E. coli RF-1 recognizes UAA and UAG, RF-2
recognizes UAA and UGA.
RF-3 binds GTP and enhances activities of RF-1
and 2.
Presence of RF with a nonsense codon at A site
transforms the peptidyl transferase into a
hydrolase, which cleaves the peptidyl chain from the
tRNA carrier
Hydrolysis of GTP is required for disassociation of
RFs, ribosome subunit and new peptide

Polypeptide synthesis

Translation in Eucaryote

Ribosome structure
-eucaryotic ribosome are larger and more complex than
procaryotic ribosome

Small & large ribosomal


subunits.

A Binding site for


the mRNA is present on
small subunit.
Two binding sites
(P & A) bind tRNAs on
large subunit.

rRNAs are believed to play a catalytic role in protein


synthesis.
After removal of 95% of the ribosomal proteins, the
60S subunit can catalyze formation of peptide bonds.
Ribosomal proteins are now believed to help fold the
rRNAs properly and to position the tRNAs.

Structure of tRNA

P site holds the tRNA


carrying the growing
polypeptide chain.
A site holds the tRNA with
the next AA to be added.

Ribosomes hold the


mRNA and tRNAs together
and connect the amino
acids at the A site to the
growing polypeptide.

Translational Initiation in Eukaryotes


Begins with methionine that is not formylated
tRNA (tRNAiMet) different from the one that is
used for internal methionine codons
Translation start determined by the AUG and
surrounding sequence
Translation start site also affected by RNA
structure at the 5 end of the mRNA

No S-D sequence.
CAP (catabolite activator protein)
binding protein (CBP) 5 end of
mRNA by binding to 5 CAP
structure
An initiation complex forms with
CBP, initiation factors and the 40S
subunit.
The complex scans mRNA looking
for the first AUG closest to the 5 end
of the mRNA
eIF-2 analogous to IF-2, transfers
tRNA to P sight. GTP hydrolysis
involed in release

Scanning (or Kozak) Model for


Translation Initiation in Eukaryotes

Scanning Model of Initiation


Proposed by M. Kozak

ATP

Small subunit of ribosome (+ initiation factors,


GTP and tRNAiMet) binds to the 5 Cap, and
scans along the mRNA until the first AUG
Translation starts at the first AUG
Model seems to work for most mRNAs
Fig. 17.16

Three distinct stages of


translation
Initiation
Rate limiting step

Sequence
of events
leading to
translation
initiation

Requires hydrolysis of ATP and GTP


Results in formation of a complex containing the mRNA, the
ribosome and the initiator Met-tRNA
A. 5 end (Cap) dependent initiation
The initiation complex binds to the 5 cap structure and
scans in a 5 to 3 direction until initiating AUG is
encountered
B. Internal ribosome entry
Initiation complex binds upstream of initiation codon

Sonenberg et al., eds., Translational Control of


Gene Expression (2000), p. 46.

5 end (cap) dependent initiation:

The first step is the recognition of the 5 cap by eIF4F, which consists
of three proteins, eIF4E, eIF4G and eIF4A.
Cap binding protein, eIF4E, binds to cap
The N-terminus of eIF4G binds eIF4E and the C-terminus binds eIF4A
The 40S subunit binds to eIF4G via eIF3

Closed loop model:

The 5 end dependent initiation is stimulated by the poly(A)


binding protein Pabp1p, which interacts with eIF4G
This interaction circularizes the mRNA and facilitates
formation of the initiation complex
Mechanism to ensure that only intact mRNA is translated

Cap-Dependent Initiation of Protein Synthesis in Eukaryotes


An initiation complex
forms at the cap with the
40S ribosomal subunit
and other translation
initiation factors.
The 40S complex then
scans down the 5
untranslated region to the
first AUG codon.
A GTP hydrolysis step by
eIF5 triggers GDP binding
of eIF2 and release of
initiation proteins.
The 60S subunit joins
the complex and the
80S ribosome initiates
translate the ORF.

Chain elongation

Aminoacyl t-RNA binding


EF-Tu undergoes a major conformational change o hydrolysing GTP
Transpeptidation
Traslocation
Translocation occurs via intermediate states
Eukaryotic elongation cycle resemble that of prokaryote

Elongation

Eukaryotic elongation process


Similar to what occurs in prokaryotes.
Analogous elongation factors.
EF-1a = EF-Tu docks tRNA in A-site
EF-1b = EF-Ts recycles EF-Tu
EF-2 = EF-G involved in translocation process

Termination
Translation is terminated
at one of three stop codons
(UAA, UAG & UGA).
Termination codon at the
A site is recognized by the
release factor instead of a
tRNA
The release factor binds
the termination codon
The peptide chain is then
released followed by
dissociation of the tRNA
and the ribosome

Ribosome selects
aminoacylated tRNA
eEF1 and GTP are
bound to
aminoacylated tRNA
Ribosome catalyzes
formation of a peptide
bond
Translocation is
dependent on eEF2 and
GTP hydrolysis
Many ribosomes may
translate mRNAs
simultaneously on the
same strand.

eEF1
GTP

P A

eEF2
GTP