Chapter twelve protein biosynthesis DNAs RNAs Proteins 1 DNA replication: entirety 2 RNA transcription: systematicness 3 protein biosynthesis: individuality.
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DNAs RNAs Proteins
1 DNA replication: entirety
2 RNA transcription: systematicness
3 protein biosynthesis: individuality
1
1
32
Concept of protein biosynthesis or translation1 The transmission of the genetic infor- mations from RNA into protein.2 Protein biosynthesis is an extraordinarily complex process in which genetic infor- mation encoded in 4 nucleotides is translated into the 20 amino acid “alphabet” of polypeptides. 3 codon-anticodon interaction
Section 1*The system of protein biosynthesis
Section 2 *Process of protein biosynthesis
Section 3 *protein post-translational processing and its transportation
Section 4
The inhibition and the interference of protein biosynthesis
The material that participates in protein synthesis
1. mRNA: the carrier of information for the assembly
2. ribosome: the site available for the assembly
3. tRNA: the carrier for the amino acids
5. enzyme and protein factor: the control factors, signallers for initiation and termination
6. free nucleotide and inorganic ion: the providers of energy for the process, ...
4. amino acids: the building blocks themselves
Mature mRNA is direct template for protein biosynthesis.
mRNA
There are a lot of mRNA in a cell.
The length of mRNA is different.
The life of mRNA is the shortest in all RNA.
mRNA
Most prokaryotic mRNAs are polycistrons (i.e. They
encode several proteins), but most eukaryotic mRNAs are
monocistrons .
The mRNA is readed in a 5’ 3’ direction during protein translation. The start codon is AUG , and stop codons are UAA, UAG and UGA in mRNA.
character of genetic codoncharacter of genetic codon
continuity/commaless:
5’ …….5’ …….A U GA U G G C AG C A G U AG U A C A UC A U …… …… U A AU A A 3’ 3’
Alanine Valine Histidine
5’…….5’…….A U GA U G G C AG C A N N G U AG U A C A UC A U …… …… U A AU A A 3’ 3’
5’…….5’…….A U GA U G G C AG C A N N N N G U AG U A C A UC A U …… …… U A AU A A 3’ 3’
5’.…….5’.…….A U GA U G G C AG C A G U AG U A C A U C A U …… …… U A AU A A 3’ 3’
5’..…….5’..…….A U GA U G G C AG C A G U AG U A C A U C A U …… …… U A AU A A 3’ 3’
degeneracy:
1x2 Methionine AUG
Tryptophan UGG
2x9 UGUCysteine UGC
3x1 AUUIsoleucine AUC AUA
4x5 GUU GUC
Valine GUA GUG
6x3 CGG CGA CGU
Arginine CGC AGG AGA
AUG
5’
3’
AUG
5’
3’
wobble:
5’ 3’ mRNAUAUUAC
Tyrosine codon
Tyrosine-tRNATyrosine-tRNA
mRNA codon base(3) A C U A G C G U
tRNA anticodon base(1) I (inosine) U C
mitochondrion chloroplast
apart from universalapart from universal
The methionine codon, tryptophan codon, start and stop codon are different with ones of nucleus in the mitochondrion and the chloroplast.
results: After a few minutes, radioactivity of protein is only in the ribosomes. After a few hours or days, radioactivity of protein is in all organelle.
conclusion: The ribosomes is the place of protein biosynthesis.
ribosome
to determine to determine radioactivityradioactivityof proteinsof proteins
radioactiveradioactiveamino acidsamino acids
the structure of ribosome in the prokaryotethe structure of ribosome in the prokaryote
small subunit
large subunit
peptidyl tRNA
5’
3’
N
new peptide strand
aminoacyl tRNA5’
3’
binding amino acid
5’ 3’
mRNA
acceptor site, A siteor aminoacyl site
peptide site, P site or donor site
5’
3’
tRNA
exit site, E site
the structure of ribosome in the eukaryotethe structure of ribosome in the eukaryote
small subunit
large subunit
peptidyl tRNA5’
3’
N
new peptide strand
aminoacyl tRNA5’
3’
binding amino acid
5’ 3’
mRNA
acceptor site, A siteor aminoacyl site
peptide site, P site or donor site
tRNA and aminoacyl tRNAtRNA and aminoacyl tRNA
5’
3’
C
C
AOH
CR
HO C O
NH2
H
Aminoacyl tRNA synthetase
ATP Mg2+
5’
3’
C
C
AO
CR
C O
NH2
H
the steps of reaction:
Amino acid + ATP + Enzyme
Aminoacyl - AMP - Enzyme + PPi
1
Aminoacyl - AMP - Enzyme + tRNA
aminoacyl-tRNA + AMP + Enzyme
2
Supplementation and explanation:
The every aminoacyl-tRNA synthetase is very specific for corresponding the amino acid and the tRNA.
OH
C
O
H
OACC
O
C
O
AMP
C
O
The aminoacyl-tRNA synthetase has a editing activity.
Supplementation and explanation:
H
OACC
OH
C
O
OH
C
O
OH
C
OO
OH
C
A amino acid can bind with 1-6 kinds of tRNA.
Supplementation and explanation:
The aminoacyl tRNA is used to write into the form as follows.
Start tRNA is abbreviated into tRNAfMet in prokaryote,
and that is tRNAiMet in eukaryote.
Arg-tRNAArg arg-tRNAargor
CH3
S
CH2
CH2
H2N CHCOO tRNAfMet
formyl transferase
+ THFA CHO
CH3
S
CH2
O CH2
HC HN CHCOO tRNAfMet
forming of fMet-tRNAfMet in prokaryote
Initiation of translation for prokaryote
The material that participates in protein biosynthesis initiation for procaryotes
IF3IF1 IF2 Three kinds of initiation factors.
GTP A nucleoside triphosphate
fMet-tRNAfMet
mRNA
30S Small
subunit
It is consist of a 16S rRNA and 21 kinds of proteins.
It is consist of 23S, 5S rRNA and 34 kinds of proteins.
50S Large subunit
Initiation of translation for prokaryote
IF3 IF1
30SIF3 IF1
50S
+
GTPIF2
+ +
fMet-tRNAfMet
Ribosome70S
30S
+GDP Pi
30SIF3 IF1
IF2GTP
IF2
IF1
IF1
Initiation of translation for prokaryote
IF3 IF1
30SIF3 IF1
50S
+
GTPIF2
+ +
fMet-tRNAfMet
Ribosome70S
30S
+GDP Pi
30SIF3 IF1
IF2GTP
IF2
IF1
IF1
Initiation of translation for prokaryote
explanation and supplementation :
The first codon in mRNA is AUG which codes for
methionine.
This AUG is called initiation codon or start codon.
Naturally, other AUG codons in the mRNA encode other
methionine residues in peptide strand.
A U GA U G
methionine methionine
A U G
methionine
Initiation of translation for prokaryote
explanation and supplementation :
There are Two different tRNA which are used to translate the
different AUG codon in mRNA respectively; the tRNAfMet is
used for the initiational codon and is called initiator tRNA
whereas the tRNAmMet is used for the internal AUG condon.
A U GA U G A U G
tRNAfMetHC
O
tRNAmMet tRNAm
MetMet Met Met
HC
O
Initiation of translation for prokaryote
explanation and supplementation :
The first amino acid of a new peptide is N-formylmethionine
(abbreviated fMet) in the prokaryotes .
It is important crucially that the initiator tRNA bind
with the start codon correctly.
CH3
S
CH2
CH2
HC — HN — CH — C —…………. HN— CH — C—OH O
O
O
R
CH3
S
CH2
CH2
H2N CHCOO tRNAfMet
Formyl transferase
+ THFA CHO
CH3
S
CH2
O CH2
HC HN CHCOO tRNAfMet
Forming of Formyl for Met-tRNAMet in prokaryote
structure of initiated complex for prokaryotic translation structure of initiated complex for prokaryotic translation
small subunit
large subunit
fMet-tRNAfMet
5’
3’
formylmethionine
5’ 3’
mRNA
acceptor site, A siteor aminoacyl site
peptide site, P site or donor site
exit site, E site
Initiation of translation for prokaryote
3’… …………………… 5’
UCCU
ribosomal binding sequence
mRNA 5’… AGGAPuPuUUUPuPuAUG……………………. 3’
Shine-Dalgarno sequence,SD sequence
rpS-1 recognizing and binding sequence
16S rRNA
ribosomesmall subunit
rpS-1 protein
Initiation of translation for eukaryote
40S Small
subunit
The material that participates in protein biosynthesis initiation for eukaryotes
60S Large subunit
It is consist of 28S, 5.8S, 5S rRNA and 45 kinds of proteins.
twelve kinds of initiation factors.
Two nucleoside triphosphate
Met-tRNAiMet Mature mRNA
It is consist of a 18S rRNA and 33 kinds of proteins.
eIF3 eIF4D
coIF2
PBAeIF4A
eIF4C
eIF4E
ATP GTP
eIF2
eIF6
eIF5
eIF4G
eIF2B
40S
GTPeIF5
Ribosome
Initiation of translation for eukaryote
eIF3
40S
eIF3
+
eIF4D
Active initiation complex
80S
+eIF4C
40S
GTP
coIF2
Met-tRNAiMet
GDP
+Pi
eIF4APAB
ATP
Pi
ADP
+
+
eIF4GeIF4E
GTPeIF2
eIF6
eIF2B
eIF2B
eIF3eIF2B
60S
40S
GTPeIF5
Ribosome
Initiation of translation for eukaryote
eIF4D
Active initiation complex
80S
+
coIF2
Met-tRNAiMet
GDP
+Pi
eIF4APAB
ATP
Pi
ADP
+
+
eIF4GeIF4E
GTPeIF2
eIF3 eIF6eIF2B
eIF4C
40S
GTP
eIF3eIF2B40S
eIF3
+eIF2B
60S
The character of initiation translation for eukaryote
It is more complicated than that of the prokaryote.
The starting Met-tRNAi Met don’t occur formylation. It requires ATP and GTP.
There is not S-D sequence in mRNA 5’-end.
The mRNAs are located correctly in the ribosome by both 5’-capping and 3’ poly A tail and assistant role of cap and poly A tail binding protein (eIF4E and PAB etc).
AUG
coding sequence
UAA
ribosome 40 s small subunit
5’ cap
3’ polyA tail
eIF-3
eIF-4E
eIF-4G polyA binding protein
mRNA
The interaction among the several initiated factors and 5’ cap and 3’ polyA tail on mRNA and ribosome small subunit in eukaryotic translational initiation stage
The elongation of translation
The material that is need in the translation elongation.
The process of translation elongation
The elongation of translation
The material that is need for thetranslation elongation
The complex of translated initiation
2 n3 The aminoacyl-tRNA
GTP A guanosine triphosphate
EF-TsEF-Tu
EF-GThe elongation factors
1
AUG
The elongation of translation
The process of elongation in translation
1. entrance
2. Peptide bond formation
3. translocation
The elongation of translation
entrance
GTPEF-TuEF-Ts + GTP
EF-Tu + EF-Ts
2
+
2 GTPEF-Tu
GTP
1
AUG
+
1
AUG
2
+EF-TuGDP
Pi
The elongation of translation
entrance
GTPEF-TuEF-Ts + GTP
EF-Tu + EF-Ts
2
+
2 GTPEF-Tu
GTP
1
AUG
+
1
AUG
2
+EF-TuGDP
Pi
The elongation of translation
formation of peptide bond in translated elongation
1
AUG
2
1
2
AUG
1 2
transpeptidasetranspeptidase
1
2+
The elongation of translation
formation of peptide bond in translated elongation
1
AUG
2
1
2
AUG
1 2
transpeptidasetranspeptidase
1
2+
The elongation of translation
translocation for translated elongation
1
2
AUG 2 3 4
EF-G
translocase
Next ribosome cycle
AUG 2 3 4
5’ 3’
1 2 3 nN C
1
2
AUG 2 3 4
The elongation of translation
translocation in translated elongation
1
2
AUG 2 3 4
EF-G
translocase
Next ribosome cycle
1
2
AUG 2 3 4
The elongation of translation
1
2
AUG 2 3 41
2
AUG 2 3 41
AUG
2
AUG 2 3 41
AUGAUG 2 3 4
entrance
peptide bond formation
translocation
Next ribosomal cycle
Process of elongation for protein translation
termination of translation
release factor (RF)
RF1
RF2
RF3
There are three kinds of release factors in termination of protein translation for prokaryote as follows.
It can recognize UAA and UAG and bind ribosome and accelerate hydrolysis of the esterbond which is between C-end of peptide strand and tRNA 3’-end.
It can recognize UAA and UGA and bind ribosome and accelerate hydrolysis of the ester bond which is between C-end of peptide strand and tRNA 3’-end.
It can bind GTP and accelerate to bind RF1 or RF2 with ribosome.
Termination of translation
the process of translation termination for prokaryote
N
1 2 3
UAA
N
1 2 3
UAARF1RF3 RF2
N
1 2 3
+
H2O
UAA
OH
RF2
UAA
OH
RF2
GTPGDP
+Pi
+
+
Next translation
IF3
IF1IF3IF1
+
Termination of translation
the process of translation termination for prokaryote
N
1 2 3
UAA
N
1 2 3
UAARF1RF3 RF2
N
1 2 3
+
H2O
UAA
OH
RF2
UAA
OH
RF2
GTPGDP
+Pi
+
+
Next translation
IF3
IF1IF3IF1
+
Termination of translation
Eukaryote:
Termination in eukaryotes is catalyzed by a single eukaryotic release factor.
All process of translation termination in eukaryote is similar to that in prokaryote in many ways.
Section 3 protein post-translational processingand its transportationThe polypeptide chain as a precursor that released from ribosome is not an active protein, which reveals bioactivity until post-translational processing has been taken.
post-translational processing
The type of post-translational processing
processing of the higher structure of protein
1 the fold of protein
2 the polymerization of subunits
3 prosthetic group binding
modification of primary structure of protein
1 N-terminal modification of new peptide chain.
2 modification of individual amino acid in peptide chain.
3 hydrolytic modification
Posttranslational processing
Polymerization of subunits
1
2
3
4
Oligomer
* Hemoglobin 22
* Transmembrane Protein * Membrane integral protein
Posttranslational processing
prosthetic group binding
1) coenzyme or enzyme contained prosthetic group
2) glycoprotein
3) lipoprotein
4) color-protein
Posttranslational processing
4
N-terminal modification
1 fMetenzyme
Met
2 fMetenzyme
prokaryote
3 Met
enzyme Met
eukaryote
Posttranslational processing
explanation
Sometimes N-terminal modification of new
peptide chain is not taken after having
terminated peptide chain synthesis, but is
following synthesis of peptide chain.
5’
N
N
C
Posttranslational processing Modification of individual amino acid in peptide chain.
C
CC
CH2
H2
H2
HN
C Oproline
hydroxylase
N
C
H2
C
CC
CHH2
HN
C O4-OH-Pro
OH
1) hydroxylation
Posttranslational processing
N C
NC
3) to form disulfide bridge
SH
HC
CN
CH2
O
Cysteine
oxidation
reductionSH
CCN
CH2
O
Cysteine
H
S
HC
CN
CH2
O
N C
NC
S
CCN
CH2
OH
Posttranslational processing
the type of disulfide bridge
(1) disulfide bridge between two peptide strands
(2) disulfide bridge in a peptide strands
Posttranslational processing
hydrolytic modification
Signal peptide Lysine
Lysine
Arginine
Arginine
pro-opio-melano-cortin, 265 aa
103 peptide 39 peptide Adreno-cortico-tropichormone
91 peptide -lipotropin
13 peptide-melanocytestimulating hormone 11 -endophin
18 peptide-melanocytestimulatinghormone
N C
5’ cap3’ poly A tail
mRNA
small subunit
large subunit
elongated peptide
ribosome
signal peptide
SRP: signal recognition particles
signal peptidase carbohydrate
ribosome
receptor
SRP receptor
SRP
secreted proteinendoplasmic reticulum and Golgi membrane system
the secretion of proteins in eukaryote
the transportation of proteins to the mitochondria in the eukaryote
Tom
Tom
Tim
Tim ATP
ADP+Pi
transmembrane tunnel
NH3+
COO-heat stroke protein 70
HSP-70++++++
______
signal sequence
out membrane in membrane
out membrane receptor complex
plasmic side
mitochondrion side
mature protein
HSP-70
the transportation of eukaryotic proteins into the nucleus
membrane
+
nuclear transportation factors
nucleoprotein
RanGTP
GDP+Pi
nuclear localizationsequence, NLS
plasmic side
nuclear matrix side nucleic pore complex
1.4.1 antibiotics
An antibiotic is not precisely definable but
is usually thought of as a product of
microbial metabolism which inhibits
growth of pathogenic organisms, without
substantially affecting any host of the
latter.
The inhibition of antibiotics to protein translation
tetracyclines
The aminoacyl - tRNA binding with ribosome in the prokaryote blocked by tetracyclines.
The tetracyclines do not readily penetrate into mammalian cells.
The inhibition of antibiotics to protein translation
chloromycetin The chloromycetin binds ribosomal large subunit of prokaryotic cell and inhibits activity of transpeptidase. When concentration of chloromycetin is higher, the chloromycetin also can block the protein synthesis of mitochondrion in eukaryotic cell.
The inhibition of antibiotics to protein translation
Streptomycin and kanamycin
They can bind ribosomal small subunit of prokaryotic
cell and change ribosomal conformation and lead to
read wrong .
The inhibition of antibiotics to protein translation
Puromycin
Puromycin is a nucleotide analagous to
Tyr- tRNATyr and so it competes with site of the
Tyr- tRNATyr in ribosome in transpeptidation
process; it combines with growing peptide but it
lacks a suitable carboxyl group and so
translation is aborted.
It is too toxic to use in eukaryotic cell , and
is a primary research tool.
cycloheximide
The cycloheximide can inhibit translocasefor eukaryote specially. Therefore it isa primary research tool.
The inhibition of antibiotics to protein translation
target site of every antibiotics for protein
translation process
tetracyclinesStreptomycin and kanamycin
chloromycetin
puromycin
cycloheximide
The inhibition of antibiotics to protein translation
Diphtheria toxin
The diphtheria toxin is a modification enzyme
The EF-2 of eukaryotes is inactivated by the
diphtheria toxin.
The inhibition of antibiotics to protein translation
interferon
The interferons include -interferon,
-interferon and -interferon.
The interferons can inhibit mainly
protein biosynthesis of the virus.
interferon viral dsRNA
2’-5’ polyA synthetase
RNase L
ATP
A
PPP
A
PPP
5’
A
2’-5’ poly A
5’
P
2’
…...
A
5’
viral mRNA decompose
2’
A 由 tRNA 某一部位上相邻的三个核苷酸组成
B 由 mRNA 某一部位上相邻的三个核苷酸组成
C 由 rRNA 某一部位上相邻的三个核苷酸组成
D 由 DNA 某一部位上相邻的三个核苷酸组成
E 由多肽链某一部位上相邻的三个氨基酸组成
5 下列关于反密码子的叙述,哪一项是正确的?
A tRNA 上的反密码子的专一性
B tRNA 的专一性
C 氨基酰 tRNA 合成酶的专一性
D rRNA 的专一性
E mRNA 上核苷酸的排列顺序
13 蛋白质生物合成中多肽链上氨基酸的排列 顺序取决于?
A AAC
B CAA
C CAC
D CCA
E ACA
15 以含有 CAA 重复序列人工合成的多核苷链 为模板,在无细胞蛋白质合成体系中能合 成三种多肽:多聚谷氨酰胺、多聚天冬酰 胺和多聚苏氨酸。已知谷氨酰胺和天冬酰 胺的密码分别是 CAA 和 AAC ,则苏氨酸的 密码是:
A 密码有种属特异性,所以不同生物合成不同蛋白质
B 密码阅读有方向性,是从 5’ 端到 3’ 端
C 一种氨基酸可以有一组以上的密码子
D 一组密码子只可以代表一种氨基酸
E 密码第三位碱基在决定所参入的氨基酸的特异性方 面作用较小
16 下列关于密码子的描述哪些项是正确的?
A 由 tRNA 识别 DNA 上的三联密码
B 氨基酸能直接与其特异的三联体密码连接
C tRNA 上的反密码能与 mRNA 上的密码形成碱基配对
D 在合成蛋白质之前,只有密码子中的全部碱基改变才 会出现由一种氨基酸替换另一种氨基酸E 核蛋白体从 mRNA 的 5’ 端向 3’ 端滑动对应着肽链从C 端 向 N 端延伸
19 蛋白质生物合成时,
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