Download Protein Biosynthesis Translation

Genetic information
transfer-----Protein Biosynthesis
Translation(翻译)
Lecturer: Jiao Li
Lecturer:
Jiao021-65986142
Li
Phone:
Phone:
021-65986142
Email:
[email protected]
Email: [email protected]
Objectives
After completion of this section, you should be able to :
1. List molecules (Aminoacyl tRNA synthetase) involved in protein
Synthesis
2. Describe the process of protein synthesis
3. Post-translational processing and destination of newly
synthesized protein.
4. Inhibitors of protein synthesis
Protein synthesis: using mRNA as the template, translate
the nucleotide sequence of mRNA into the amino acid sequence
of protein according to the genetic codon.
Replication
Transcription
Translation
Outline of the section
1. System of protein biosynthesis
2. Process of protein translation
3. Post-translational modification
4. Inhibitors of protein synthesis
Section 1
Protein Biosynthesis
System
Protein synthetic System

Direct template— mRNA

tRNA

Ribosomes
●
20 amino acids (AA)
●
Enzyme and protein factors: IF、eIF、EF、RF
●
ATP、GTP、Mg2+
Characteristics：complicated, accurate,
dynamic,quick, energy consumed（90%）
§1.1 Genetic codon carrier-mRNA
● base
amino acid conversion：Genetic code
a three-nucleotide codon in a nucleic acid (mRNA)
sequence specifies a single amino acid, initiatior codon or
terminator codon. This kind of three-nucleotide codon is called
genetic codon or triplet codon.
Initiator codon: AUG
Terminator codon: UAA、UAG、UGA
Standard Genetic codon
Salient features
Sense codon：code for amino acid — 61.
Nonsense codon：code for no amino acid— terminator
codon—UAA，UAG，UGA
Initiator codon ：as initiation signal for translation —
AUG, GUG(prokaryote) .
A complete sequence of mRNA, from the initiation
codon (AUG) to the termination codon, is termed
as the open reading frame (ORF), which codes for
the primary structure of polypeptide.
ORF
5 '
AU G
UTR
3'
U A A
UTR
 Properties
of genetic code
1. Commaless
The genetic codons should be read continuously
without spacing or overlapping.
× spacing
UUCUCGGACCUGGAGAUUCACAGU
UUCUCGGACCUGGAGAUUCACAGU
※ Accuracy of start site
×
overlapping
Gene damage may causebase insertion or deletion
on mRNA template, which leads to frame shift
mutation.
Insertion
Deletion
2. Sideness
Reading frame：5’—3’
Translation ：5’—3’
3. Degeneracy
code1
amino acid
(except Trp, Met)
Code2/3/4/6
Providing multiple options for maintaining stabilization
of species.
AA
Number of genetic
codon
AA
Number of genetic
codon
4. Universal
• The genetic codons for amino acids are always
the same. （basic reason of microbial infection）
• With a few exceptions ： mitochondrial mRNA,
chloroplast .
Differences between mitochondrial codons and
standard codons
Codon
Normal sense
Mitochondria
AUG、AUU、AUA
AGA、AGG
UGA
Ile
Arg
Terminal codon
Initiation codon
Terminal codon
Trp
§1.2 Amino acid carrier—tRNA/adaptor
AA arm
DHU loop
TC loop
Anticodon loop
“Cloverleaf ” structure of tRNA
●
tRNA is the bridge between protein
and genetic data
tRNA 2
Amino
acid
Iso-tRNA
tRNA 3/4/6
relative specificity of transferring
Binding sites in tRNA
O
3'
O
C
H
C
R
NH2
Attachment site of aa
DHU loop
(binding rRNA in
ribosome)
A—C—G
mRNA —U—G—C
Mathch with codons
摆动性(wobble base pair)
Non-Watson-Crick base pairing
the 5' base on the anticodon, which binds to the 3'
base on the mRNA, was not as spatially confined as the
other two bases, and could, thus, have non-standard
base pairing.
wobble
U
Wobble hypothesis
1
mRNA:
2
3
G C U
G C C
5’ —G—C—A——3’
tRNA ： 3’
—C—G—I——5’
3
2
1
Anticodon of ala
from yeast
The first base of anticodon is key to determine the number of
codons it can identify.
Diagram of wobble base pairing
5’base on anticodon
3’base on codon
A
U
C
G
G
U
C
U
A
G
U
I
C
A
●
Activated amino acid —aminoacyltRNA
Aminoacyl-tRNA：gly-tRNAgly
Initiation aminoacyl-tRNA： (f)met-tRNAi(f)met
Elongation aminoacyl-tRNA： ala-tRNAeala
§1.3 Protein synthesis site:
（Ribosomes）
Components of ribosome
Prokaryote
S
Eukaryote
riboso
me
Small
subunit
Large
subunit
riboso
me
Small
subunit
Large
subunit
70S
30S
50S
80S
40S
60S
rRNA
16SrRNA
5S-rRNA
23S-rRNA
18SrRNA
28S-rRNA
5S-rRNA
5.8S-rRNA
protein
rpS
21kinds
rpL
36 kinds
rpS
33kinds
rpL
49 kinds
rRNA
rRNA is important component in ribosome and
plays a key role in ribosome assembling and
functioning.
1. Maintaining structure of ribosome. Delete rRNA
and ribosome will collapse.
2. Involved in binding to mRNA.
3. Key component in protein synthesis.
Illustration of protein synthesis in prokaryote
Small
subunit
P site (Peptidyl site)
D/donor site
A site
(Aminoacyl site)
Acceptor site
E site
(Exit site)
Nascent
peptide
chain
AA
Large
subunit
Function of ribosome in protein synthesis
① Containing protein-conducting channel
② Containing binding sites of IF, EF and RF
③ Containing three RNA binding sites， （ donor site, D 位);
( peptidyl site , P 位）; （ acceptor site, aminoacyl site , A 位）
④ Peptidyl transferase activity
⑤ GTPase activity
Section 2
The Process of Protein
Biosynthesis
Protein synthesis process
Amino acid activation
Ribosome cycle
§2.1 Activation and transferration of amino acid
a. Aminoacyl-tRNA synthetase
aa + tRNA
Aminoacyl-tRNA synthetase
aminoacyl- tRNA
ATP
AMP＋PPi
Diagram of aminoacyl-tRNA synthetase
tRNA
ATP
aminoacyl-tRNA synthetase
Step 1 of reaction
aa＋ATP-E —→ aminoacyl-AMP-E ＋ PPi
～
～
A
AA
～
A
Gain high
energy
Aminoacyl-AMP
Step 2
aminoacyl-AMP-E
＋
tRNA
↓
aminoacyl-tRNA
＋
AMP
＋
E
A
Adenosine
Aminoacyl-AMP
Aminoacyl-tRNA synthetase
A
Aminoacyl-tRNA
Activation of aa and
attachment to tRNA
Generation of
amino-acyl tRNA
catalyzed by
amino-acyl tRNA
synthetase
b. Features of aminoacyl-tRNA synthetase

1. high specificity to substrate-amino acid.

2. proofreading activity，second code
system—tRNA。
Match correct aa to its
corresponding tRNA
§2.2 Ribosome cycle（prokaryote）
Protein synthesis initiates from AUG at 5’
end and decipher genetic codons to polymerize
aa residues until stop codon occurs.
Translation process：

Initiation

Elongation

Termination
Major
phases
Idetification of initiation site — SD sequence
Prokaryote
mRNA
5′
3′
ORF
UTR
Ribosomal binding site
Initiation codon
UTR
mRNA
nt
16S-rRNA
S-D sequence
（ribosomal binding site）
1. Initiation
Translational initiation complex = mRNA +
initiation aminoacyl-tRNA + ribosome
substrate + initiation factor + GTP + Mg2+
substrate + initiation factor + GTP + Mg2+
stage pro
IF1
IF2
function
Binding small subunit 30S
Binding initiation amino-acyltRNA to AUG, GTPase
IF3
Binding small subunit
30S,facilitate the dissociation
of ribosome
Formation of translational initiation
complex in prokaryote
（1）Ribosome acitivation
（2）30S- initiation complex
（3）70S- initiation complex
（1）Ribosome activation
IF-1
IF-3
（2）30S-initiation complex
a. mRNA binds with small subunit of ribosome
5'
SD
sequence
3'
AUG
IF-1
IF-3
b. Initiation aminoacyl-tRNA( fMet-tRNAimet ) binds to small
subunit of ribosome
Formyltransferase
IF-2 GTP
5'
SDsequence
3'
AUG
IF-1
IF-3
（3）70S-initiation complex
Large subunit binds to form 70s initiation complex
GTPPi
IF-2 GDP
5'
SDsequence
3'
AUG
IF-1
IF-3
Pi
GTP
IF-2 -GTP
GDP
5'
SDsequence
IF-3
3'
AUG
IF-1
2. Elongation
The second and the next aminoacyl-tRNA in
line bind to the ribosome along with GDP and
elongation factor.

It consists of the following steps:
• (entrance/registration)
• (transpeptidation)
• (translocation)
Initiation complex + elongation factor + GTP + Mg2+
Elongation Factors required for peptide
polymerization
Elongation
factor
function
EF-Tu
Transferring aminoacyl-tRNA to A site,
GTPase activity
EF-Ts
Replace GDP to bind Tu
EFG
Translocase activity
（1）Entrance
(registration)
Initiating
codon
Correct aminoacyl-tRNA
enters A site.
Ternary
complex
Second
code
Aminoacyl-tRNA
EF-T functions in
entrance (prokaryote）
Tu/Ts cycle
Initiating
codon
Second
code
Amino-acyltRNA
Enter quickly
GTP
Tu Ts
Ts
Tu GDP
5'
AUG
GTP
3'
（2）Peptide bond formation
Transpeptidase catalyze the formation of peptide
bond.
Large subunit：rRNA + rproteins
（3）Leave（E site）
（4）Translocation: EF-G has translocase activity,
which catalyzes movement of peptidyl-tRNA from A
site to P site.
EF-G
fMet
Transpeptidase
fMet
Tu GTP
5'
AUG
EF-G
3'
Peptide chain elongation
3. Termination
When the A site of the ribosome faces a stop codon
(UAA, UAG, or UGA), no tRNA can recognize it, but
releasing factor can recognize nonsense codons and
causes the release of the polypeptide chain.
Newly synthesized peptide chain is released and the
translational machine dissociate.
Release factors are involved in termination
(release factor, RF)
RF in prokaryote: RF-1，RF-2，RF-3
Function of RF
RF-1
recognizing UAA, UAG
RF-2
recognizing UAA, UGA
RF-3
GTP binding protein and helps to
convert transpeptidase to esterase.
Termination in
prokaryote
transpeptidase
IF3
esterase
COORF
5'
UAG
3'
Energy and ions needed
Amino acid activation：2
Synthesis process：4( initiation, entrance,
translocation, release)
Inorganic ions：Mg2+, K+
(Polyribosome)
a cluster of ribosomes, bound to a mRNA
molecule and read one mRNA
simultaneously, progressing along the mRNA
to synthesize the same protein.
Direction of
translation
——Protein synthesis with
high speed and efficiency.
Electron micrograph of protein
synthesis indicates polyribosome
§2.3 Protein synthesis in eukaryote
— different from prokaryote
More factors and more steps
1. Translation is uncoupled with transcription
2. 5' cap and 3' poly(A) tail are involved in
initiation of protein translation.
3. No SD sequence in mRNA
4. Ribosome
1. Prokaryote: translation is coupled with transcription
转录
翻译
Eukaryote: uncoupled gene expression
transcription
细胞核
processing
Nuclear membrane
细胞质
translation
2. Prokaryote (Polycistron)
5 PPP
3
protein
Eukaryote (monocistron)
① Necessary for
entering cytoplasm
② Increase stability
尾
3AAA
5 mG - PPP
cap
protein
① Protection from
damage of nuclease
② Binding small subunit
Noncoding sequence
SD sequence
③ Involved in initiation
of translation
Coding seuquece
Start codon
Stop codon
3. Biological synthesis process
A. initation
(1) More factors
(2) Different binding sequence
40S
60S
①
eIF-1、eIF-3
60S
40S
elF-3
②
met
Met
Met-tRNAiMet-elF-2 -GTP
mRNA
ATP
③
(elF4E, elF4A, elF4G, );elF4B
ADP+Pi
= elF4E
PAB
mRNA
elF4E
elF4E: Cap binding protein
PAB
Met
PAB: Poly A tail binding protein
Release of IFs
elF-5
④
Met
GDP+Pi
1. Met-tRNAiMet-elF-2 –GTP bind
2. mRNA is recognized
small subunit——43S initiation
complex
3. mRNA join—48S initiation
complex ，Scan for AUG
4. Large subunit join—80S
initiation complex, IF relaease
B. Elongation in eukaryote
Similar to prokaryote but with different factors
EF-Tu —— eEF1α
EF-Ts ——eEF1βγ
EF-G ——eEF-2
There is no E site in eukaryote.
C. Termination in eukaryote：one RF
4. Protein synthesis in mitochondria and chloroplast is similar to
that in prokaryote.
Factors involved in translation
stage pro
eu
function
IF1
IF2
eIF2
Involved in formation of initiation complex
IF3 eIF3、eIF4C
Initia
CBP I (eIF4E)
eIF4A B F
Scan for AUG
eIF5
GTPase
eIF6
EF-Tu
Elong
Facilitate release of 60s subnuit
eEF1
Assist entrance
EF-Ts
eEF1 
Help recycling of EF-Tu, eEF1
EF-G
eEF2
Translocase
RF-1
Term
Binding cap of mRNA
RF-2
Binding stop codon
eRF
Release of peptide chain
Section 3
Posttranslational Processing &
Protein Transportation
To obtain their final structural features and
related function, newly formed polypeptides
undergo various forms of modifications.
Including:
* Primary structure modification
* Advanced structure modification
§3.1 Primary structure modification
a. Modification of N-terminus
Removing of (f)met-tRNAi(f)met/ N-terminal sequence
aminopeptidase
b. Covalent modification
1. Hydroxylation （collegen）
2. Glycosylation
（globular protein）
3. Phosphorylation（regulation）
4. Acetylation （universal）
5. Carboxylation
（clotting factor）
6. Methylation
7. Ubiquitinoylation （for degredation）
8. Esterification （membrane-bound protein）
Hydroxylation
Collegen
Gly - Pro - X （ X can be any aa）
OH
Hydroxyproline, hydroxylysine—
—post-translational modification
Case1：disease resulted from incorrect
post-translational modification
（Scurvy）
Cause：Vit C deficiency
Vit C（coenzyme of
hydroxylase）
（pro，lys）hydroxylase
hydroxylation of collagen
assemble and
stablization of collagen
Carboxylation（羧化）
Carboxylation of thrombinogen（凝
血酶原）
HOOC-CH- CH２- CHCOO
HOOC
＋
－
NH3
γ –carboxyglutamic acid ——
chelating Ca2+
Case2： disease resulted from incorrect posttranslational modification
coagulopathy- vit K deficency
Vit K epoxide reductase
vit K cycle
γ –hydroxylation of
coagulation factor
c. Proteolytic modification（
*Removing of
N-terminal signal peptide
* Activiation of zymogen
* One peptide chain is hydrolyzed to
many active peptides
d. Formation of
disulfide bond
Active
center
Processing of proinsulin
C peptide
HS
HS
SH
C From
chain A
SH
HS
After removal of
signal peptide,
From
proinsulin folds into
chain B
correct conformation
Signal peptide
N
S－S
N
S
S
C
S
S
proinsulin
SH
preproinsulin
C peptide is further
cleaved and muture
insulin is formed.
N
N
C
S
S
S
S
insulin
A chain
C
B chain
§3.2 Modification of advanced
structure
a. Folding of newly formed peptide chain
b. Subunit polymerization (obtaining function)
c. Coenzyme connection（glycoprotein,
lipoprotein,conjugated enzyme）
a. Polypeptide chain folds into natural
conformation
* Correct secondary structure, motif, domain
and final conformation are formed
stepwisely.
* Primary structure is the basis of advanced
structure
* In the present of accesary molecules: enzyme
or chaperon.
Molecules involved in newly polypeptide folding
1. (molecular chaperon)
2. (protein disulfide isomerase, PDI)
3. (peptide prolyl cis-trans isomerase, PPI)
•Molecular chaperon
Molecular chaperones are proteins that assist the non-covalent
folding/unfolding and assembly/disassembly of other macromolecular
structures, but do not occur in these structures when the structures are
performing their normal biological functions having completed the
processes of folding and/or assembly.
1. (heat shock protein, HSP)
HSP70, HSP40 and HSP90 family
2. (chaperonins)
Prokaryote: GroEL（eu: HSP60）and GroES(HSP10)
family
Mechanism of action of HSP as chaperon
HSP70 assist folding of protein by cycle of binding and
dissociating from hydrophobic segment of protein.
HSP40 binds
nascent peptide
HSP70-ATP complex
Hydrolysis of ATP
HSP40- HSP70-ADP-complex
ATP
ADP
Dissociate and peptide is released to fold correctly
Protect newly peptide chain from aggregation,
misfolding and inhibit “off path-way” folds.
Chaperonin
Providing a microenvironment for protein to fold
GroEL/GroES
Protein folding after translation
Properties of chaperon
1. There is no specificity to nascent polypeptide chain
2. Coupled with hydrolysis of ATP
3. Contain no information about particular folding patterns.
4. Multiple functions.
5. Conserved in primary sequence.
§3.3 Destination of newly synthesized
protein
• (protein targeting)
Protein is targeted to destination
(cytoplasm, membrane and extra-cell
area).
• (signal sequence)
Protein contains signal sequence(usually
in N-terminus) which target protein to its
destination.
Signal sequence for protein destination
protein
Secretary
Signal sequence
Signal peptide
protein
Protein in ER
Signal peptide，C terminal-Lys-Asp-Glu-LeuCOO-(KDEL sequence)
Protein in mt
N terminal portion（20～35氨基酸残基）
Protein in
（-Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val-，SV40 T
nucleolus
antigen）
Protein in
-Ser-Lys-Leu-（PST sequence）
peroxisome
Protein in
Man-6-P
Target of secreted protein
• (signal peptide)
The conserved sequence in Nterminus of secreted protien.
Primary structure of signal peptide
Basic aa
N-terminus
hydrophilic
hydrophobic aa
hydrophobic area
Splicing site
Splicing site
（signal recognition particle，SRP）
SRP composition：6 proteins+ 7S RNA
SRP function：bind SRP receptor(docking protein，DP
） on ER.
Signal peptide directed protein to ER
Secretary protein synthesis pathway
(biological reactor)
Clone cow
Transgene animal ： biological reactor—
—“animal pharmceutical factory”
Coagulation factor, albumin
Section 4
Interference & Inhibition of Protein
Biosynthesis
cycloheximide
chloromycetin
puromycin
tetrocyclin
Streptomycin and kanamycin
Many antibiotics work because they inhibit
protein synthesis in bacteria
purom
ycin
Anolog of tyrosyl-tRNA
mechanism of
transpeptidase
puromycin
•(diphtheria toxin)
C
O
EF-2
NH2
+
ADP
O
CH2
N
+
O
（active）
diphtheria
OH
C
OH
O
EF-2
ADP
NH2
N
O
CH2
O
（inactive）
+
OH
OH
Mechanism of interferon
1. Interferon induce phosphorylation of eIF2
Interferon activate eIF2 kinase
dsRNA
ATP
eIF2
Pi
ADP
eIF2-P（inactiveblock translation）
phosphatase
2. Interferon induce degredation of
virus RNA
interferon
A
A
dsRNA
PPP
PPP
2’-5’A synthase
5
ATP
A
A
2
2
P
P
5
5
2- 5A
RNaseL
RNaseL
active
Degrade mRNA
Summary
1. Many protein factors are involved in translation.
2. Process of protein translation is still divided into
initiation, elongation and termination.
3. Newly synthesized proteins need to be further
Post-translational modified to have correct
structure and perform function.
4. Inhibitors of protein synthesis like antibiotics are
usually used to treat diseases in clinic.