Download Chapter 9 .Metabolism of nucleotide

Chapter 9 .Metabolism of nucleotide
Section 1. Metabolism of nucleotide
1.the synthesis of purine nucleotide :
de novo synthesis
salvage pathway
※ de novo synthesis: The pathway which uses phosphoribose (R-5-P),amino acid ,one
carbon unit and CO2 as materials and goes through a series of enzyme-catalized reactions to
synthesize purine nucleotide is called de novo synthesis .
※ salvage pathway: The pathway which uses free(ready-made) purine or purine nucleoside
as materials and goes through simple reactions to synthesize purine nucleotide is called
salvage pathway .
(1) the course of de novo synthesis:
1 IMP synthesis:
○
※ Characteristic: Purine nucleotide is synthesized stepwise on the phosphorobose (R-5-P)
molecule rather than synthesizing purine base along fristly and then binding to
phosphoribose.
※ From “sweet” to “bitter”
(2) salvage pathway:
APRT
Adeline (reade-made) + PRPP
AMP+Ppi
HGPRT
Inosine (reade-made) + PRPP
I MP+Ppi
HGPRT
Guanine (reade-made) + PRPP
GMP+Ppi
Lack of HGPRT induce Lesch-Nyhan Syndrome.
(3) the sythesis of deoxyribonucleotide:
NMP+ATP
NDP+ADP
Ribonucleotide reductase
NDP
dNDP
(4) antimetabolism of purine nucleotide:
6MP(6-mercaptopurine) is similar to
inosine
azaserine
is similar to
Gln
aminopterin , MTX
is similar to
FH
2.Catabolism of purine nucleotide :
AMP
Isosine
Xanthosine
Uric acid ※
GMP
Adenine
Excessive uric acid induce Gout. We use allopurinol to treat the Gout in clinic because
allopurinol is similar to inosine.
Section 2. Metabolism of pyrimidine nucleotide
1.the synthesis of pyrimidine nucleotide :
de novo synthesis
salvage pathway
(1) de novo synthesis:
※
CPSⅡ(it locate in cytoplasm)
CPSⅠ(it locate in Mit) (urea cycle)
CO2+Gln
carbamoyl phosphate
characteristic:
※ Pyrimidine nucleotide is synthesized stepwise purine base along fristly and then binded to
phosphoribose rather than synthesizing pyrimidine stepwise on the phosphorobose (R-5-P).
※ From “bitter” to “sweet”
(2)salvage pathway:
pyrimidine+PRPP
pyrimidine nucleotide
PPi
2.antimetabolism:
5-Fu(fluorouracil)
is similar to
3.catabolism:
Cytosine
Thymine.
Thymine
Uracil
CO2+NH3
H2N-CH2-CH2-COOH
β- Ala
H2N-CH2-CH-COOH
CH3
β-aminoisobutyric acid ※
Department 3. Transfer of genetic information
Gene : Gene is DNA functional fragment encoding biological active products which mainly are
protein , various RNA.
The central dogma:
transcription
replication
DNA
translation
RNA
protein
reverse transcription
RNA
Chapter 11. Replication (the biosynthesis of DNA)
Replication is a process in which genetic information is transmitted from parental
DNA to daughter DNA molecules.
Section 1. Semi-conservation replication
1.concept : The original double-stranded DNA opens up and both strands serve as template for
the synthesis of new DNA. The products of the reaction are two daughter double
–stranded DNA molecules each of which has one original template strand and one
strand of newly synthesized DNA.
2.experimental evidence :
F0
F0
F1
F2
3.the signification of semi-conservation: fidelity
F1
F2
Centrifugation
Section 2. Enzymes in DNA replication
1. the reaction of replication: (dNMP)n+ dNTP
(dNMP)n+1
+
PPi
or
dAMP-dTMP –dGMP + dCTP
dAMP-dTMP -dGMP- dCMP +PPi
2. DNA polymerase (DNA-dependent DNA polymerase, DDDP):※
(1) reactive properties:
a. dNTP as substrate
b. single strand DNA as template
c. free 3’-OH as primer
d. synthetic direction of new strand is 5’
3’
(2)In prokaryote cells:
polymerase
a. Polymerase Ⅰ: single peptide chain, the function is proofreading and repairing.
1 DNA polymerase (5’→ 3’) activity: fill the gap caused by exonuclease Polymerase
○
activity recognizes the next deoxynucleotide on the DNA template and then adds a
complementary dNTP to the 3’-OH of the primer ,creating a 3’,5’phosphodiester bond and
releasing PPi(pyrophosphate).
2 3’→5’exonuclease activity:it can remove mismatched deoxynucleotide
○
starting from the 3’end of a chain
3 5 ’ →3’exonuclease activity:it can hydrolyze nucleic acid primer
○
starting from the 5’end of a chain
Proteinase hydrolyze polymerase Ⅰ into two fragment, larger one and smaller one .The larger
one has the activity of DNA polymerase activity and 3’→5’exonuclease activity, also called
Klenow fragment ,which can be use as “tool enzyme” in molecular study.
b. polymerase Ⅱ:only has its activity without DNA-pol Ⅰand Ⅲ
c. polymerase Ⅲ:It is the most effective polymerase in synthesis of new strand DNA. It consist
of 10 subunit .αεθ subunit consist of core enzyme
1 DNA polymerase (5’→ 3’) activity:
it has
○
2 3’→5’exonuclease activity
○
(3). In eukaryote cells: DNA polymerase α β γ δ ε
DNA pol α: extend the lagging strand
DNA pol Ⅲ
DNA pol δ: extend the leading strand
locate in nucleus
DNA pol ε: is similar to DNA pol Ⅰ
DNA pol β: only has its activity without other enzyme
DNA pol γ: exist in Mit
locate in Mit
(4). Exonuclease and fidelity:
1 exonuclease:
○
Polymerase Ⅰ degrade DNA in two terminus, so it has the activity of
3’-exonuclease and 5’-exonuclease which can remove the mismatch deoxynucleotide
and primer RNA. DNA pol Ⅰalso has the activity of polymerase, so it can fill the gap
caused by exonuclease.
2 Proofreading: The course that polymerase Ⅰremove the mismatch deoxynucleotide
○
and then add the correct deoxynucleotide to the gap are called proofreading
3
○ fidelity and base select:
Dependence of DNA polymerase on template ,make the parent DNA and daughter
DNA matched precisely, confirm correct transfer of genetic information.
Fidelity of DNA is dependent on 3 mechanisms:
a. absolute base paired regulation
b. base selective function of polymerase on DNA elongation
c. proofreading function when mistake happened in DNA replication
3.helicase and topoisomerase:
(1)helicase: Unwinding of double helix DNA is required before replication, because the template
must paired with its complement dNTP. Helicase can use the ATP to unwind double helix
DNA forming a replication fork.
Three mainly kinds of helicase:
a.DnaA :recognize the origin of replication
b.DnaB :true helicase, open double helix
helicase
c.DnaC :cooperate with DnaB
(2)topoisomerase: During unwinding double helix DNA, the downstream double helix DNA over
warpped. If DNA over wraped ,called positive-superhelix ,and if wrapped not enough ,called
negative-superhelix. There are two kinds of topoisomerase:
It can break phosphodiester bond of just one strand of the duplex and then rejoin it, thus
unwind supercoiled (positive-superhelix) DNA.
DNA topoisomerase break the double helix , unwind supercoiled DNA(when ATP
presence).
positive-superhelix
negative-superhelix
positive-superhelix
(topoisomerase Ⅰ)
(topoisomerase Ⅱ)
(3)SSB(single-stranded DNA binding protein):When DNA was unwinded into two single
strand ,if paired base existed, it intend to form double-stranded DNA again.SSB binds to single
strand DNA and keep the single state of DNA in replication.
+
DNA
SSB
4.primase and primosome
(1) primase:※
The replication is initiated by the formation of a short RNA(approximately five
nucleotides long) which served as the primer for DNA polymerize. The synthesis of this
short fragment RNA was catalized by primase , using DNA as template , so primase is
DNA dependent DNA polymerase (DDDP).
Primer provide the free3ˊ-OH for the polymerize of the first deoxyribonucleotide .
(2) primosome
Complex of Dna A. B. C , replicative factors , binding to DNA template , called
primosome .
Why primer is RNA instead of DNA ? DNA polymerase can not catalyze the polymerize
of two free deoxyribonucleotide ,but RNA polymerase can.
5. DNA ligase:
During replication , the synthesis of new strand may be discontinuous , the DNA ligase
can link two adjacent terminus by 3’-5’phosphodiester bond .
It is the “tool enzyme” in genetic engineering.
5ˊp
3ˊ-OH
5ˊp
3ˊ-OH
ATP
DNA ligase
ADP
O
‖
5ˊp
O — P — O
3ˊ-OH
|
O-
Section 3 .The course of biosynthesis of
DNA
Replication is a continuous process , to describe it clearly , we separate it into three stage :
initiation , longation , termination artificially .
1. initiation of biosynthesis
First step : helicase unwind of double helix DNA forming a replication fork.
It include:
DnaA recognize the origin of replication
DnaB : open double helix
DnaC :cooperate with DnaB
Second step: topoisomerase unwind supercoiled (positive-superhelix) DNA
Third step: SSB binds to single strand DNA and keep the single state of DNA in replication.
The forth step: primase synthesize primer RNA (provide the free3ˊ-OH)
concept:
1) bi-directional replication
in prokaryote cell , initiation started in single ori C site , extending in two direction , called
bi-directional replication .
origin C
2) replication fork
the double helix opens up , and both single strands served as template for synthesis of new
DNA , forms “Y” shape , called replication fork .
3)replication bubble (replication eye ) : the region of replicating DNA associated with the
single origin is called replication bubble . each replication bubble consists of two replication
forks moving in two opposite direction .
DNA synthesis proceeds until the replication bubble merge together .
4) replicon
in eukaryote cell , many origins form many replication bubbles at the same time , the DNA
replicated under the control of single origin is called a replicon .
replicon
(20-80 units)
replication unit
5)replication unit : the replication origins are activated in clusters , called replication unit ,
consisting of 20-80 origins .
6) primosome: original region together with helicase , Dna C protein , primerase , the
complex is called primosome .
7) leading strand and lagging strand※
Double strand DNA is antiparallel , one strand runs 5ˊ-3ˊ and the complementary
strand runs 3ˊ-5ˊ.
As the original double-stranded DNA opens at a replication fork , new strand is made
against each template strand .Therefore , on the template strand with 3ˊ-5ˊ orientation ,
new DNA is made in a continuous piece in the correct 5ˊ-3ˊ direction , called leading
strand .
On the other template strand (that has 5 ˊ -3 ˊ orientation) , DNA polymerase
synthesizes short pieces of new DNA in 5ˊ-3ˊ direction , and the joins these pieces
together by ligase . The new strand which is made by this discontinuous method is called
lagging strand .
5’
helicase
5’
3’
5’
3’
lagging strand
okazaki fragment
SSB
Leading strand
5’
3’
2.elongation
the chemical essence, is the formation of phosphodiester bond .
the enzyme catalyze this reaction , is DNA-pol Ⅲ in prokaryote cell , and DNA-polαδ in
eukaryote cell .
(1) DNA-pol δ : catalyze the formation of long new strand DNA or catalyze the
continuous replication in leading strand .
DNA-polα : catalyze the formation of short new strand within hundreds of nucleotides
or catalyze the discontinuous replication in lagging strand .
(2) DNA-pol Ⅲ catalyze synthesis of both strand , which is carried out by different subunit .
(3) okazaki fragment※
In the process of replication , the synthesis of leading strand is continuous , in lagging
strand , DNA polymerase synthesize short pieces of new DNA in 5ˊ-3ˊ direction , and
then joins them together . The discontinuous fragments in lagging strand are called
okazaki fragment .
(4) rolling circle replication
Special form of DNA replication in simple life-form (plasmid) , or DNA outside of
chromosome.
At first , a nick show in one strand , 5ˊ- terminal stretch out . single strand serve as
template , the synthesis of new strand is discontinuous .the other one has no nick , can
replicating when circling , the synthesis of new strand is continuous .
No primer is needed . 3’
3’OH
5’
3’
3. termination of replication
5’
when replication finished , the 5ˊ- terminal of okzaki fragment is RNA instead of DNA ,so
this primer must be cut out , and DNA should fill it , then join these fragment together at
last . Mmany kinds of enzymes involved in this process :
1) RNase hydrolyze RNA primer, gap show between fragments
2) DNA-pol Ⅰ catalyze the DNA synthesis in gap , until it was filled up , not DNA-pol Ⅲ
DNA-pol Ⅰuses its 5ˊ-3 ˊexonuclease activity to remove the RNA primer and then fills
the gap with new DNA
3) DNA ligase
when the new synthesis DNA reachs the next fragment , it is stop. The two free terminals
form a nick , DNA ligase joins them using ATP .
3’
5’
5’
3’
RNase or pol Ⅰ(5ˊ-3 ˊexonuclease activity)
3’
5’
5’
3’
5’
3’
pol Ⅰ( polymerase activity)
3’
5’
5’
5’
3’
DNA ligase
3’
5’
5’
4. telomere and telomerase in eukaryite cell
(1) telomere
Telomere is a structure at the terminus s of linear DNA molecule in eukaryote cell .
It is important for chromosome to maintain its stability and integrity , also called “bio-clock”.
Telomere is the G. C – rich repeated nucleotide sequence .
(2) telomerase ※
Telomerase is the complex of RNA and protein . The protein is RNA dependent DNA
polymerase , it is a kind of reverse transcriptase . The RNA is template. So telomerase
can add DNA repeats(six deoxynucleotide) to the end of eukaryotic chromosome .
Section 4
Impair and repair of DNA
Mutation ( DNA damage ) : the change of structure , replication and phenotype in one or more
deoxyribonucleotide , is called mutation .
that is , the change of genetic materials cause the change of genetic information .
1 significance of mutation
(1) foundation of evolution & differentiation
(2) no phenotype change mutation
(3) lethal mutation
(4) cause of genetic disease
2 factors for mutation
(1)spontaneous mutation rate : 10 –9
(2) induced mutation
1) physical factor
a. UV ( ultra violet ) : cause convalent combination of adjacent pyrimidine in
DNA chain , form pyrimidine dimer . eg: thymine dimer
b. radiation & radiator : X-ray , isotope
2) chemical factor
a. base analog : 5-BU
b. hydroxylamine
c. nitrite
d. alkylating agent eg : nitromins
3 type of mutation ※
(1) mismatch (point mutation) , transition and transversion
(2) deletion , insertion and frame-shift mutation
(3) rearrangement : large fragment exchange in DNA molecule
transposition & exchange recombination
4 DNA damage repairing
(1) light repairing
(2) excision repairing: most important repairing mechanism in cell .
mutation
indonuclease
5’
pol Ⅰ
5’
3’
pol Ⅰ
ligase
5
3’
(3) recombination repairing
5’
3’
5’ 3’
5’ 3’
5’
3’
5’ 3’
(4) SOS repairing
Section 5 .Reverse transcription & reverse transcriptase
※Reverse transcription is a process in which genetic information is transmitted from
RNA to DNA .
Reverse transcriptase is a kind of RDDP .
reaction process :
reverse transcriptase
RNase
integration
RNA template
duplex
double strand DNA
integration : when virus infects the living cell , it can join host genome by recombination in
some cases , and replicate and express with host cell .
integration is a important way for virus to lead cancer , which can not integrate into host
without being turned to double helix by reverse transcription
retrovirus : RSV HIV .
Chapter 12. Transcription (biosynthesis of RNA)
Concept:
※ Transcription is a process in which genetic information is transmitted from selected DNA
to RNA .
※
The mode of transcription is asymmetric transcription. It has two meanings:
First, in the double strand DNA, one strand is copied, but the other strand is not.
Second, the strand being copied is not always in the same strand, or sometimes one
strand is copied, sometimes the other.
※
The single strand DNA being copied is called template strand or Watson strand.
The other strand that is not copied is called coding strand or Crick strand. It has the
same sequence as the transcriptive product RNA, except that the RNA contains U instead
of T.
Compare of transcription with replication:
1. Sameness:
Template:
DNA
Enzyme:
polymerase
Synthesis direction:
5’ → 3’
2. Difference:
Replication
Transcription
Mode
Semi-conservation replication
Asymmetric transcription
Template
Both strands of DNA
Single strand of DNA
Substrate
dNTP
NTP
Base pairing
A-T C-G
A-U A-T C-G
Enzyme
DDDP or DNA polymerse
DDRP or RNA polymerase
Primer
Need
Don’t need
Produce
DNA
RNA
Section 1. Template and enzymes.
1. Template : The template of transcription is the single strand DNA
template strand
RNA
coding strand
DNA
structural gene
coding strand
template strand
2. Enzyme: transcriptase ( RNA polymerase) or DDRP(DNA directed RNA polymerase)
(1)In prokaryote cells:
RNA pol from prokaryotes is a large multisubunit enzyme , consisting of five
subunits ,which are twoαsubunits, oneβ,one β’and oneσsubunit.
The subunit of RNA pol
Function
α
Decide which gene to be transcripted
β
catalyzed the polymerization of RNA
β’
Bind DNA template and then unwind the DNA
locally to expose a single-strand template
σ
Recognize the promoter site
concept :The complete RNA pol (include two α-subunits, oneβ,one β’and oneσ
-subunit) is called the holoenzyme(α2ββ’σ). RNA pol without the σ-subunit is
called core enzyme(α2ββ’).
※ Although core enzyme has catalytic function, it cannot initiate transcription specifically
at promoter sites; only the complete holoenzyme can do this.
Clinic significance: rifampicin can combine with β’-subunit and inhibit transcription.
(2). In eukaryote cells: RNA polymerase ⅠⅡ Ⅲ
※
RNA pol
RNA pol Ⅰ
The product
45S-rRNA
RNA pol Ⅱ
hnRNA
RNA pol Ⅲ
5S-rRNA,tRNA ,snRNA
3. The recognizing and binding of template and RNA pol
※promoter is the DNA consensus sequence which RNA pol recognized and binded.
Two important prompter consensus sequence are found in prokaryote , one located about
10 nucleotides (-10 sequence)upstream of where transcription will begin and one located
about 35 nucleotides up stream(-35 sequence). The consensus sequence TATAAT located in
–10 sequence is also called Pribnow box. ※
Pribnow box
DNA
-35
-10
5’
TTGACA
TATAAT
3’
promoter
promoter
Transcriptional start site
Section 2.The course of transcription
The RNA produced by transcription are primary transcripts which have activities
through processing.
1. Initiation
(1) In prokaryote cells: ※
RNA polymerase holoenzyme (α2ββ’σ) initiates transcription. This involves
recognition of the –10 sequence (Pribnow box) and –35 sequence, and then unwinds
the double strand DNA to expose a single strand DNA template that can be copied.
Transcription initiation complex=RNA pol(α2ββ’σ)-DNA-pppGpN-OH3’
(2) In eukaryote cells:
The initiation of RNA synthesis by polymerase is directed by the presence of
promoter site on the 5’ side of the transcriptional start site. The promoter consensus
sequence is TATA, also called Hogness box or TATA box. There are other promoters
upstream the transcriptive start site, such as CAAT box and GC box , these boxes
belong to cis-acting element(it is DNA sequence). But the proteins recognizing and
binding the cis-acting element are necessary for the initiation .The proteins belong
to trans-acting factor. The trans-acting factor which can bind RNA pol directly or
indirectly are called TF(transcriptional factor).
2. Elongation
Following initiation ,theσ-subunit dissociates from RNA pol to leave the core
enzyme (α2ββ’) that continues RNA synthesis in a 5’
3’ direction using NTP as
precursors.
The RNA pol ,DNA template and RNA transcript form a transcription bubble or
transcription complex.
3. Termination
In prokaryote cells:
(1). rho(ρ) factor dependent termination※
It allow recognition of the termination site and stop transcription
(2).non-ρ factor dependent termination:
Transcription continues until a termination signal is reached. The simplest
termination signal is a GC-rich region that a palindrome, followed by an AT –rich
sequence. The RNA made from the DNA palindrome is self-complementary and so
base-pairs internally to form a hairpin structure followed by a few U residues.
UU
hairpin
5’
GCCGCCAG
C
CGGCGGUC
3’ HO-UUUUA
GG
In eukaryote cells:
Section 3. Post-transcriptional modification (in eukaryote cell)
1. mRNA : include splicing , capping and polyadenylation ※
hnRNA
mRNA
RNA pol Ⅱ catalyze hnRNA synthesis
(1) RNA splicing:
Concept:
# Most structural genes in prokaryote are discontinuous, also called splite
gene.
# The coding sections of the gene(called exons ) are interrupted by
noncoding sections of DNA(called introns) .
# RNA splicing is the precise cutting out of intron sequences and joining the
ends of neighboring exons to produce a functional mRNA molecule.
# In the course of RNA splicing, introns becomes branched at a point to
form a lariat RNA.
# RNA splicing requires the involvement of small nuclear RNA(snRNA).
snRNA that has catalytic activity is also called ribozyme(an RNA enzyme).
(2) Capping of mRNA
Immediately after synthesis , the 5’ end of the primary transcript is
modified by the addition of a methylated guanine cap.
(3) polyadenylation
Next, the 3’ end of the primary RNA is added a poly(A) tail (about
100-200 A residues).
The endonuclease cleaves the RNA transcript at a site of a AAUAA
sequence that is called a polyadenylation signal. Poly(A) polymerase then adds
100-200 A residues to the new 3’ end using ATP as precursor.
2. rRNA : 45S-rRNA 5S-rRNA
rRNA
RNA pol Ⅰ catalyze 45S-rRNA synthesis
RNA pol Ⅲ catalyze 5S-rRNA synthesis
Most eukaryote have more than 100 copies of these genes that encode rRNA.
The genes for 18S , 5.8S and 28S-rRNA are typically clustered together and
tendemly repeated which are interrupted by untranscribed spacer DNA.
The 45S-rRNA is cleaved to release the 18S , 5.8S and 28S-rRNA molecules.
Processing may involve snRNA.
18S-rRNA + protein
small subunit of ribosome
5S-rRNA
28S-rRNA
+ protein
large subunit of ribosome
5.8S-rRNA
ribosome is the synthesizing factory of protein.
3. tRNA:
RNA pol Ⅲ catalyze tRNA
synthesis
Four different types of RNA processing steps
# the 5’ extra sequence is cleaved off by ribonuclease P.
# splicing introns that are always located in the anticodon loop
# add CCA-OH to the 3’end of Trna
# about 10% of the base in tRNA are modified, for example methylation (A→
mA), reduction(U → DHU), conversion of uridines to pseudo-uridine and
deamination(A → I).※