Translation

Chapter 12 The synthesis of protein molecules

using mRNA as the template, in other
word, to translate the nucleotide

T
Translation
l i sequence off mRNA
RNA into
i t the
th amino
i acid
sequence of protein according to the
id

genetic codon.

The biochemistry and molecular

biology department of CMU

The protein folds

Gene Expression to form its
working shape

Gene
DNA
Section 1
Cell machinery
CELL copies the code
G T A C T A making an mRNA
The order of bases in molecule. This

Chromosome
NUCLEUS DNA is a code for
making proteins. The
moves into the
cytoplasm.
Protein Synthetic System
code is read in groups of
Ribosomes read the
three
code and accurately
AUGAGUAAAGGAGAAGAACUUUU join Amino acids
together to make a
M S K CACUGGAUA
E E L F T protein
3

1
Protein synthesis requires multiple
elements to participate and coordinate.

• mRNA, rRNA, tRNA

• substrates: 20 amino acids
• Enzymes and protein factors:
initiation factor (IF), elongation
factor (EF), releasing factor (RF)
• ATP, GTP, Mg2+

§1.1 Template and Codon polycistron

5-PPP 3
• Messenger RNA is the template for
the protein synthesis. protein
• Prokaryotic mRNA is polycistron,
monocistron
that is, a single mRNA molecule may
3
code for more than one peptides. 5-mG - PPP

• Eukaryotic mRNA is monocistron, protein

that is, each mRNA codes for only
Non-coding ribosomal protein binding site
one peptide. Stop codon
Coding region Starting code

2
 Genetic codon

• Three adjacent nucleotides in the 5´-

3´ direction on mRNA constitute a
genetic codon, or triplet codon.
• One genetic codon codes for one
amino acid.

Genetic codon
• Three codons for stop signal:
UAA, UAG, UGA.
• One codon for start signal: AUG.
It also codes for methionine.
methionine
• 61 codons for 20 amino acids.

3
 Properties of genetic codon commaless

1. Commaless The genetic codons should be read

A complete sequence of mRNA, from continuously without spacing or
the initiation codon to the termination overlapping.
codon is termed as the open reading
codon, spacing

frame.

ORF

5' AU G UAA 3'

overlapping

Frameshift 2. Degeneracy

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• Except Met and Trp, the rest amino 3. Wobble
acids have 2, 3, 4, 5, and 6 triplet Non-Watson-Crick
codons. base pairing is
• More than one codon can specify permissible
the same amino acid between the third
nucleotide of the
• These degenerated codons differ codon on mRNA
only on the third nucleotide. and the first
GCU ACU nucleotide of the
GCC
Ala
ACC
Thr anti-codon on
GCA ACA
GCG ACG
tRNA.

Base-pair of codon and anticodon 4. Universal

The genetic codons for amino acids
are always the same with a few
exceptions of mitochondrial mRNA.

Cytoplasm Mitochondria

• AUA: Ile • AUA: Met, initiation

• AUG: Met, initiation • UGA: Trp
• UAA, UAG, UGA: • AGA, AGG:
termination termination

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§1.2 tRNA and AA Activation Activation of amino acid
tRNA

Activated amino acid

Ala-tRNAAla
Ser-tRNASer
Met-tRNAMet

6
 Summary of AA activation Protein synthesis fidelity

• Active form • Aminoacyl-tRNA synthetase has the

aminoacyl－tRNA proofreading ability to ensure that
the correct connection between the
• Activation site AA and its tRNA.
-
 carboxyl group
• It recognizes the incorrect AA,
• Linkage cleaves the ester bond, and links the
ester bond correct one to tRNA.
• Activation energy
2 high-energy bonds

Prokaryotic Met-tRNAmet Initiation tRNA

• Prokaryotic Met-tRNAmet can be For prokaryotes:
formylated to fMet-tRNAimet.
• fMet-tRNAimet can only be recognized
by initiation codon.
Met-tRNAmet + N10-formyl tetrahydrofolate
• Met-tRNA
Met tRNAemet is used for elongation
elongation.
formyl
transferase For eukaryotes:
• Met-tRNAimet is used for initiation.
fMet-tRNAimet + tetrahydrofolate • Met-tRNAemet is used for elongation.

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§1.3 Ribosomes Molecular components of ribosome
of prokaryotes
• Ribosome is the place where protein
synthesis takes place.
• A ribosome is composed of a large
subunit and a small subunit, each of
which is made of ribosomal RNAs
and ribosomal proteins.

Ribosome of prokaryotes Three sites on ribosomes

location function

Aminoacyl site Composed by Accepting an

(A site) large and small aminoacyl-tRNA
aminoacyl tRNA
subunit

Peptidyl site Composed by Forming the

(P site) large and small peptidyl bonds
subunit
Exit site Only on large Releasing the
(E site) subunit deacylated tRNA

8
 A site, P site and E site

General concepts
• The direction of the protein
synthesized : N-terminal→C-terminal
Section 2
• The direction of template mRNA: 5´→
3´end

Protein Synthetic Process • The process of Protein :

initiation
elongation
termination

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§2.1 Initiation Shine-Dalgarno (S-D) sequence
Prokaryotic initiation
• Four steps:
– Separation between 50S and 30S
subunit
– Positioning mRNA on the 30S subunit
– Registering fMet-tRNAimet on the P site
– Associating the 50S subunit
-AGGA PuPuUUUPuPu AUG-

• Three initiation factors: IF-1, IF-2 and • purine rich of 4-9 nts long
IF-3. • 8-13 nts prior to AUG

Alignment of 16S rRNA Initiation 1-2

• The IF-1 and IF-3
bind to the 30S
subunit, making
separation
between 50S and
30S subunit.

The 3´end of 16s rRNA has consensus • The mRNA then

sequence UCCU which is complementary binds to 30S
subunit.
to AGGA in S-D sequence (also called
ribosomal binding site).

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 Initiation 3 Initiation 4

• The 50S subunit

• The complex of combines with this
the GTP-bound complex.
IF-2 and the
• GTP is hydrolyzed
fMet-tRNA
to GDP and Pi.
enters the P
site. • All three IFs depart
from this complex.

eukaryotic initiation

Pi
IF-2 -GTP
GTP
GDP
• Four steps:
5' 3'
AUG – Separation between 60S and 40S
IF-3
IF-1
IF 1 subunit
b it
– binding Met-tRNAimet on the 40S subunit
– Positioning mRNA on the 40S subunit
– Associating the 60S subunit
One GTP is consumed in initiation
course。

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 Eukaryotic initiation factors
Factor Function
Facilitates binding of initiating Met-tRNAMet to 40S ribosomal
eIF2
subunit
eIF2B, eIF3 First factors to bind 40S subunit; facilitate subsequent steps
RNA helicase activity removes secondary structure in the
eIF4A mRNA to permit binding to 40S subunit; part of the eIF4F
complex
Binds to mRNA; facilitates scanning of mRNA to locate the
eIF4B
first AUG
eIF4E Binds to the 5’ cap of mRNA; part of the eIF4F complex
Binds to eIF4E and to poly(A) binding protein (PAB); part of
eIF4G
the eIF4F complex
Promotes dissociation of several other IFs from 40S subunit
eIF5 as a prelude to association of 60S subunit to form 80S
initiation complex
Facilitates dissociation of inactive 80S ribosome into 40S
eIF6
and 60S subunits

40S elF--3
elF
mRNA §2.2 Elongation
② ATP
Met elF4E, elF4G,
Met ③ elF4A, elF4B,PAB
Met-tRNAiMet-elF-2 -GTP
• Three steps in each cycle:
ADP+Pi
– Positioning an aminoacyl-tRNA in the A
60S site--- Entrance
– Forming
F i a peptide
tid bond---Peptide
b d P tid bond b d
Mett
Me
eIF-2B、
eIF-2B、eIF
eIF--3、 elF-5 elFs formation
① ④
eIF-6 GDP+Pi – Translocating the ribosome to the next
40S codon---Translocation

• Elongation factors (EF) are required.

60S Met Process of
eukaryotic initiation

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 Step 1: Entrance

• An AA-tRNA occupies the empty A site.

• Registration of the AA-tRNA consume
one GTP.
• The entrance of AA-tRNA needs to
activate EF-T.

Step 2: Peptide bond formation

GTP
Tu Ts
• The peptide bond formation occurs at
the A site.
Ts
• The formylmethionyl group is
Tu GDP transferred to α–NH2 of the AA-tRNA
5' AUG 3'
at the A site by a peptidyl transferase.

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 Peptide bond formation 1 Peptide bond formation 2

Step 3: Translocation Translocation

• EF-G is a translocase.
• GTP bound EF-G provides the energy
to move the ribosome one codon
toward the 3’ end on mRNA
mRNA.
• After the translocation, the
uncharged tRNA is released from the
E site.

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 Eukaryotic elongation

• Elongation factors are EF-1 (EF-T)

and EF-2 (EF-G).
fMet

fMet • There is no E site on the ribosome.

Tu GTP

5' AUG 3'

§2.3 Termination Termination 1

• Prokaryotes have 3 release factors:

RF-1, RF-2 and RF-3.
– RF-1 and RF-2: Recognizing the
termination codons
– RF-3: GTP hydrolysis and coordinating
RF-1/RF-2 and rpS
• The peptidyl
• Eukaryotes have only 1 releasing transferase is
factor: eRF. converted to an
esterase.

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 Termination 2

• The uncharged
tRNA, mRNA, and
RFs dissociate COO-
from the
ribosome. RF
5' UAG 3'

Translation of prokaryotes Polysome

Proteins are synthesized on a single

strand mRNA simultaneously, allowing
highly efficient use of mRNA.
mRNA

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§3.1 Protein Folding
• The macromolecules assisting the
formation of protein secondary
structure include
– molecular
l l chaperon
h individual domains and whole
– protein disulfide isomerase (PDI)
– peptide prolyl cis-trans isomerase (PPI)
Section 3
Protein Modification and
Protein Targeting
Chaperons
• A group of conserved proteins that
can recognize the non-native
conformation of peptides and
promote the correct folding of
peptides.
• Heat shock protein (HSP)
HSP70, HSP40 and GreE family
• Chaperonin
GroEL and GroES family
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 Mechanism Mechanism

• Protect the unfolded segments of

peptides first, then release the
segments and promote the correct
folding.
• Provide a micro-environment to
promote the correct native
conformation of those peptides that
cannot have proper spontaneous
folding.

§3.2 Modification of primary §3.2 Modification of spatial

structure structure

• Removal of the the first N-terminal • Assemble of subunits: Hb

methionine residue
• Attachment of prosthetic groups:
• Covalent modification of some amino glycoproteins
acids (phosphorylation, methylation,
• Connection of hydrophobic aliphatic
acetylation, …)
chains
• Activation of peptides through
hydrolysis

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§3.4 Protein Targeting
• The correctly folded proteins need to
be transported to special cellular Section 4
compartments to exert desired
biological functions.
• AAs sequence on the N-terminus that Interference of Translation
directs proteins to be transported to
proper cellular target sites is called
signal sequence.

Antibiotics
• The protein synthesis is highly
regulated. streptomycin and
karamycin Tetracycline
• This process can also be the primary
target for many toxins, antibiotics and 55' 33'
interferons.
P site A site
• These interferants interact specifically
with proteins and RNAs to interrupt
Puromycin
the protein synthesis. chloromycetin
cycloheximide

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 Antibiotics Puromycin
name target function • It has a similar
tetracycline 30S block the A site to prevent structure to Tyr-
binding of AA-tRNA with 30S
tRNA.
streptomycin 30S repress the translocase
• It works for both
chloromycetin 50S block the peptidyl transferase, prokaryotes and
and inhibit the elongation
cycloheximide 60S repress the translocase,
eukaryotes.
inhibit the elongation
puromycin ribosome of P release the prematured
and E peptide
Erythromycin 50S Inhibit the translocase

Toxins
• Ricin
• among the most
toxic substance
known
• The size of few
grains of table
salt can kill an
adult human
• acts on 60s
subunits.

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