Transcript Ribosome binding site Polysomes （多聚核糖体）

Chapter 8
Protein synthesis
Protein synthesis
• Aspects of protein synthesis
• Mechanism of protein synthesis
(Prokaryotic)
• Initiation in eukaryotes
• Translational control and posttranslational events
8.1: Aspects of
protein synthesis
•
•
•
•
•
Codon-anticodon interaction
Wobble （变偶性）
Ribosome binding site
Polysomes （多聚核糖体）
Initiators tRNA
Codon-anticodon
interaction
In the cleft of the
ribosome, an antiparallel formation of
three base pairs
occurs between the
codon on the mRNA
and the anticodon on
the tRNA.
Some highly purified tRNA
molecules were found to interact with
more than one codon, and this ability is
correlated with the presence of
modified nucleosides in the 5’anticodon position, particularly inosine
（次黄嘌呤）(formed by posttranscriptional processing of
adenosine by anticodon deaminase)
Wobble
To explain the redundancy of the genetic
code. 18 aa are encoded by more than
one triplet codons which usually differ at
5’-anticodin base
5'-anticodon base is
able to undergo more
movement than the
other two bases and
can thus form nonstandard base pairs as
long as the distances
between the ribose
units are close to
normal.
All possible base pairings at the wobble
position
No purine-purine or pyrimidine-pyrimidine
base pairs are allowed as ribose distances
would be incorrect (Neat!).
U is not found as 5’-anticodon base
Wobble pairing: non Waston-crick
base paring
Ribosome binding site
(Shine-Dalgarno sequence)
• Solely for prokaryotic translation
• A purine-rich sequence usually
containing all or part of the sequence
5'-AGGAGGU-3'
• Upstream of the initiation codon in
prokaryotic mRNA
• To position the ribosome for
initiation of protein synthesis
Shine-Delgarno element
Polysomes
• Each mRNA transcript is read
simultaneously by more than one ribosome.
• A second, third, fourth, etc. ribosome starts
to read the mRNA transcript before the first
ribosome has completed the synthesis of
one polypeptide chain.
• Multiple ribosomes on a single mRNA
transcript are called polyribosomes or
polysomes.
• Multiple ribosomes can not be positioned
closer than 80 nt.
Polysomes (多聚核糖体)
• Electron micrographs of ribosomes actively
engaged in protein synthesis revealed by
"beads on a string" appearance.
核糖体是蛋白质合成的部位
• 放射性同位素标记氨基酸注射小鼠，取肝脏制备
亚细胞器，发现微粒体中放射性强度最高，证明
核糖体是蛋白质合成的场所。
• 核糖体存在的形式：
基本类型
附着核糖体
游离核糖体
70S的核糖体
80S的核糖体
主要成分
r蛋白质：40%，核糖体表面
rRNA:60%,，核糖体内部
11-14
1 rRNA
• 小亚基: 16s RNA在识别mRNA上的多肽合成起始位
点时起重要作用；SD sequence.
• 大亚基: 23s RNA存在一段与tRNAMet序列互补的
片段。在23s RNA靠近5‘’端处，有一段序列（143
－157nt）与5s RNA（72－83）结合。
• 5s RNA有两个保守区，一个有保守序列CGAAC,与
tRNA上TC环相互作用。另一个保守序列与23s
RNA互补。这是小亚基和大亚基的相互作用。
11-15
核糖体小亚单位rRNA
(a) E.coli 16S rRNA；（红色为高度保守区）
(b) 酵母菌18S rRNA，它们都具有类似的40个臂环结构(图中1～40)，其长度和位
置往往非常保守；P、E分别代表仅在原核或真核细胞存在的rRNA的二级结构。
(Darnell et al.，1990)
2 核糖体蛋白
• 原核生物
• 30s 小亚基有21种蛋白，16s rRNA;
• 50s 大亚基有36种蛋白，5s和23s rRNA;
• 真核生物
• 40s 小亚基有33种蛋白，18s rRNA;
• 60s 大亚基有49种蛋白，5s,5.8s, 28s
rRNA;
11-17
原核生物与真核生物核糖体成分的比较
11-18
3 核糖核蛋白复合体
E.coli （a）核糖体小亚单位中的部分r蛋白与rRNA的结合位点）
（b）及其在小亚单位上的部位
（引自Albert et al.，1989，图a; Lewin,1997,图b
11-19
4 原核生物的核糖体
11-20
5 真核生物的核糖
体
11-21
Initiator tRNA
• Methionine (甲硫氨酸)is the first
amino acids incorporated into a
protein chain in both prokaryotes
(modified to N-formylmethionine) and
eukaryotes.
• Initiator tRNAs are special tRNAs
recognizing the AUG (GUG) start
codons in prokaryotes and
eukaryotes.
• Initiator tRNAs differ from the one that
inserts internal Met residues.
Initiator tRNA, fMet-tRNAfMet in E. coli
Lacking alkylated A endorses more
flexibility in recognition in base pairing (both AUG and GUG).
Initiator tRNA formation in
E. coli
1. Both initiator tRNA and noninitiator tRNAmet
are charged with Met by the same methionyltRNA synthetase to give the methionyl-tRNA
2. Only the initiator methionyl-tRNA is modified
by transformylase to give N-formylmethionyltRNAfmet.
First step:
ATP links to AA
+ H
- P P P
H3N C COO
R
+ HO
H3N C C-O- P
O
+
OH OH
Amino acid
R
AARS
OH OH
ATP
Second step: Aminoacyl-AMP reacts with tRNA
+ HO
H3N C C-O- P
Aminoacyl-AMP
O
O
+
R
OH OH
OH OH
Aminoacyl-AMP
O
tRNA
P P
AARS
O
+
O
C
C
OH
O
+
H 3N C H
R
Aminoacyl-tRNA
P
O
OH OH
AMP
原核细胞中起始氨基酸活化后，还要甲酰化，
形成甲酰蛋氨酸tRNA，由N10甲酰四氢叶酸提
供甲酰基。而真核细胞没有此过程。
运载同一种氨基酸的一组不同tRNA称为同
功tRNA。一组同功tRNA由同一种氨酰基tRNA合
成酶催化。氨基酰tRNA合成酶对tRNA和氨基酸
两者具有专一性，它对氨基酸的识别特异性很
高，而对tRNA识别的特异性较低。
8.2: Mechanism of protein
synthesis (Prokaryote)
Protein synthesis falls into three stages .
1.initiation-the assembly of a ribosome on
an mRNA molecule.
2.elongation-repeated cycles of amino acid
addition.
3.termination-the release of the new protein
chain.
Initiation
In prokaryotes, initiation requires
• the large and small ribosome
subunits,
• the mRNA
• the initiator tRNA
• three initiation factors .
Size comparisons show that the ribosome is large
enough to bind tRNAs and mRNA.
IF1 and IF3 bind to a
free 30S subunits.
IF2 complexed with GTP
then bind to the small
subunits, forming a complex
at RBS.
The initiator tRNA can then
bind to the complex at the P
site paired with AUG codon.
30S initiation complex
The 50S subunits can now
bind. GTP is then hydrolyzed
and IFs are released to give
the 70S initiation complex
The assembled
ribosome has two
tRNA-binding sites,
which are called A- and
P-site, for aminoacyl
（氨酰基位点） and
peptidyl sites（肽酰基
位点） respectively.
Only fMet-tRNAfMet
can be used for
initiation by 30S
subunits; all other
aminoacyl-tRNAs are
used for elongation by
70S ribosomes.
Elongation
With the formation of the 70S initiation
complex, the elongation cycle can begin.
Elongation involves the three factors, EFTu, EF-Ts, EF-G, as well as GTP,
charged tRNA and the 70S initiation
complex.
The three steps of elongation
1.Charged tRNA is delivered as a complex with
EF-Tu and GTP .（进位反应）
2.Peptidyl tranferase （肽酰基转移酶）(50S
ribosomal subunit) makes a peptide bond by
joining the two adjacent amino acid without the
input of more energy. （转肽反应）
3.Translocase (EF-G), with the energy from GTP,
moves the ribosome one codon along the
mRNA, ejecting the uncharged tRNA and
transferred the ribosome peptide from the
mRNA. （移位反应）
EF-Tu-Ts exchange cycle
Peptide bond
formation takes
place by reaction
between the
polypeptide of
peptidyl-tRNA in
the P site and the
amino acid of
aminoacyl-tRNA
in the A site.
Translocation
• In bacteria, the discharged tRNA leaves the
ribosome via another site, the E site.
• In eukaryotes, the discharged tRNA is
expelled directly into the cytosol.
• EF-G (translocase) and GTP binds to the
ribosome, and the discharged tRNA is ejected
from the P-site in an energy consuming step.
• the peptigly-tRNA is moved from A-site to Psite and mRNA moves by one codon relative to
the ribosome
P-site
E-site
A-site
Translocation in E. coli
Termination
Protein factors called release factors interact with
stop codon and cause release of completed
polypeptide chain.
RF1 and RF2
recognizes the
stop codon
with the help
of RF3
The release factors
make peptidyl
transferase transfer
the polypeptide to
water, and thus the
protein is released
Release factors
and EF-G:
remove the
uncharged tRNA
and release the
mRNA,.
8.3: Initiation in eukaryotes
Most of the differences in the mechanism of
protein between prokaryotes and eukaryotes
occur in the initiation stage, where a
greater numbers of eIFs and a scanning
process are involed in eukaryotes.
The eukaryotic initiator tRNA does not
become N-formylated.
真核细胞蛋白质合成的起始
真核细胞蛋白质合成起始复合物的形
成中需要更多的起始因子参与，因此起
始过程也更复杂。
（1）需要特异的起始tRNA即，tRNAfmet，并且不需要N端甲酰化。已发
现的真核起始因子有近10种（eukaryote
Initiation factor,eIF）
（2）起始复合物形成在mRNA5’端AUG
上游的帽子结构，（除某些病毒mRNA外
）
（3）ATP水解为ADP供给mRNA结合所需
要的能量。真核细胞起始复合物的形成
过程是：翻译起始也是由eIF-3结合在
40S小亚基上而促进80S核糖体解离出60S
大亚基开始，同时eIF-2在辅eIF-2作用
下，与Met-tRNAfmet及GTP结合，再通过
eIF-3及eIF-4C的作用，先结合到40S小
亚基，然后再与mRNA结合。
mRNA结合到40S小亚基时，除了eIF-3参
加外，还需要eIF-1、eIF-4A及eIF-4B并
由ATP小解为ADP及Pi来供能，通过帽结
合因子与mRNA的帽结合而转移到小亚基
上。但是在mRNA5’端并未发现能与小亚基
18SRNA配对的S-D序列。目前认为通过帽
结合后，mRNA在小亚基上向下游移动而
进行扫描，可使mRNA上的起始密码AUG在
Met-tRNAfmet的反密码位置固定下来，
进行翻译起始。
prokaryotic
Initiation factor
IF1 IF3
IF2
Elongation factor
EF-Tu
EF-Ts
EF-g
eukaryotic function
eIF3 eIF4c
eIF6 eIF4B
eIF4F
eIF2B eIF2
eIF5
Bind to ribosome submits
Bind to mRNA
Initiator tRNA delivery
Displacement of other
factors
eEF1α
eEF1βγ
eEF2
Aminoacyl tRNA delivery
Recycling of EF-Tu or
eEF1α
Translocation
Termination factors
RF1, RF2, RF3
Polypeptides Chain release
eRF
Scanning
The eukaryotic 40s ribosome submit
complex bind to the 5’cap region of
the mRNA and moves along it
scanning for an AUG start codon.
Eukaryotic
ribosomes migrate
from the 5’ end of
mRNA to the
ribosome binding
site, which includes
an AUG initiation
codon.
Initiation
In contrast to the events in prokaryotes,
initiation involves the initiation tRNA
binding to the 40S subuits before it can bind
to the mRNA. Phosphorylation of eIf2,
which delivers the initiation tRNA, is an
important control point.
The initiation factor can be grouped to there
function as follow
Binding to ribosomal
subunits
eIF6 eIF3 eIF4c
Binding to the mRNA
eIF4B eIF4F eIF4A
eIF4E
Involved in initiation
tRNA delivery
eIF2 eIF2B
Displace other factors eIF5
Initiator
eIF3+4C+
tRNA+eIF2+GTP 40S
Ternary
complex
+
43S ribosome
complex
43S preinitiation complex
ATP
ADP+Pi
+mRNA+eIF4F
+eIF4B
48S preinitiation
complex
Scanning
More factors
involved
Scanning to
find AUG
Elongation
The protein synthesis elongation cycle in
prokaryotes and eukaryotes is quite
similar.
The factors EF-Tu EF-Ts EF-G have direct
eukaryotic equivalents called eEF1α
eEF1βγ eEF2
Termination
Eukaryotes use only one release factors eRF,
which requires GTP,recognize all three
termination codons.
Termination codon is one of three (UAG,
UAA, UGA) that causes protein synthesis
to terminate.
8.4: Translational control
and post-translational events
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Translational control
Polyproteins
Protein targeting
Protein modification
Protein degradation
Translational control
• In prokaryotes, the level of translation of
different cistrons can be affected by: (a)
the binding of short antisense molecules,
(b) the relative stability to nucleases of parts
of the polycistronic mRNA ,
(c) the binding of proteins that prevent
ribosome access.
In eukaryotes,
1. protein binding can also mask the mRNA
and prevent translation,
2. repeats of the sequence 5'-AUUUA -3' can
make the mRNA unstable and less
frequently translated.
Polyprotein
• A single translation
product that is cleaved
to generate two or more
separate proteins is
called a polyprotein.
Many viruses produce
polyprotein.
Protein targeting
• The ultimate cellular location of proteins is
often determined by specific, relatively
short amino acid sequence within the
proteins themselves. These sequences can
be responsible for proteins being secreted,
imported into the nucleus or targeted to
other organelles.
Prokaryotic protein targeting:
secretion
Eukaryotic protein targeting
• Targeting in eukaryotes
is necessarily more
complex due to the
multitude of internal
compartments:
• There are two basic
forms of targeting
pathways
1.
2.
The secretory pathway
in eukaryotes (co-translational targeting)
• The signal sequence of secreted
proteins causes the translating
ribosome to bind factors that make
the ribosome dock with a membrane
and transfer the protein through the
membrane as it is synthesized.
Usually the signal sequence is then
cleaved off by signal peptidase.
Protein modification
• Cleavage:
– To remove signal
peptide
– To release mature
fragments from
polyproteins
– To remove internal
peptide as well as
trimming both Nand C-termini
• Covalent modification:
– Acetylation;
– Hydroxylation;
– Phosphorylation;
– Methylation;
– Glycosylation;
– Addition of nucleotides.
Phosphorylation
Protein degradation
• Different proteins have very different halflives. Regulatory proteins tend to turn over
rapidly and cells must be able to dispose of
faulty and damaged proteins.
Protein degradation: process
Faulty and damaged proteins are attached
to ubiquitins (ubiquitinylation).
The ubiquitinylated protein is digested by a
26S protease complex (proteasome) in a
reaction that requires ATP and releases intact
ubiquitin for re-use.
• In eukaryotes, it has been discovered that the
N-terminal residue plays a critical role in
inherent stability.
– 8 N-terminal aa correlate with stability:
Ala Cys Gly Met Pro Ser Thr Val
– 8 N-terminal aa correlate with short t1/2:
Arg His Ile Leu Lys Phe Trp Tyr
– 4 N-terminal aa destabilizing following chemical
modification:
Asn Asp Gln Glu