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From Human to DNAFrom Human to DNA
GenesGenes
....TTGGAATTTTCCGGGGTT
AAAATTGGAACCAAGGTT....
CellsCells
ChromosomChromosom
eses
DNADNA
HumaHuma
nn
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1 2 3 45 UTR 3 UTR
From ChromosomeFrom Chromosome
to Geneto Gene
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NUCLEOTIDES are the subunits of nucleic acid,
Including DNA. Each of these subunits is made up of:
AN ORGANIC BASE
A PENTOSE SUGAR
Note that the phosphate group
is bonded to the C-5 atom of the
pentose sugar
NH2
C H
N C
C C
O N H
O-
-O P O CH2 O
O- C C
H HH H
C C
OH H
1
23
4
5
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STRUCTURE OF THE NUCLEIC ACIDSSTRUCTURE OF THE NUCLEIC ACIDSSTRUCTURE OF THE NUCLEIC ACIDSSTRUCTURE OF THE NUCLEIC ACIDS
NUCLEIC ACIDS ARE POLYMERS OF NUCLEOTIDES
- Deoxyribonucleic acid (DNA)
- Ribonucleic acid (RNA)
NUCLEOTIDE STRUCTURE
DNA and RNA, are polymers of nucleoside
monophosphates (nucleotides) group.
Each nucleotide consists of :
1. A Pentose sugar :
a. Ribose sugar RNAb. Deox ribose su ar DNA
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2. A Nitrogenous base :2. A Nitrogenous base :
a. Purines : adenine (A) and guanine (G) found in DNAor
RNA
b. Pyrimidins : Cytosine (C), thymine (T), and uracil (U)
Thymines are found only in DNA
Uracils are found only in RNA
3. Phosphate
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The normal base-pairing arrangement found in DNA.The normal base-pairing arrangement found in DNA.(The dashed lines indicate hydrogen bonds)(The dashed lines indicate hydrogen bonds)
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THE SECONDARY STRUCTURE OF DNATHE SECONDARY STRUCTURE OF DNATHE SECONDARY STRUCTURE OF DNATHE SECONDARY STRUCTURE OF DNA
THE DOUBLE HELIX
1. Two antiparallel strands form a right-handed helix
2. Complementary base pairing
a b
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TranscriptionTranscription
Transcription
1 2 3 4
Start (ATG) Stop (TGA, TAA, TAG)
Genomic DNA
5 UTR 3 UTR
1 2 3 4
Start (AUG) Stop (UGA, UAA, UAG)
Primary RNA
5 UTR 3 UTR
2 copies
many
copies
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(1) Multiple factor and RNA polymerase II are needed to initiate
transcription from TATA box promoters. Including RNA polymeraseII, more than 40 different polypeptides are needed to initiate
transcription
(a) Transcription factor IID (TFIID) recognizes and binds to the
TATA box sequences independently of RNA polymerase II
(i) The TATA box-biding protein (TBP) is the subunit of
TFIID that binds to the TATA-box DNA sequence
(ii) TFIID is made of eight other subunits called TBP-asso ciated
factros (TAF)
(b) Five other transcription factors are required for the proper initation
of transcription by RNA polymerase II (TFIIA, TFIIB, TFIIF,
TFIIE AND TFIIH)
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There is no poly (T) sequence
in the DNA template that
corresponds to this tail
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SplicingSplicing
Primary RNA
1 2 3 4
Start (AUG) Stop (UGA, UAA, UAG)
5 UTR 3 UTR
2 315 UTR 4 3 UTRmRNA
Start Stop
splicing2 3
2 3
GT AGIntron 2
exons
introns
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Primary RNA
1 2 3 4
Start (AUG) Stop (UGA, UAA, UAG)
5 UTR 3 UTR
2 315 UTR 4 3 UTRmRNA
315 UTR 4 3 UTRmRNA
215 UTR 4 3 UTRmRNA
15 UTR 4 3 UTRmRNA
Splicing:Splicing:
FlexibilityFlexibility
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TranslationTranslation
2 315 UTR 4 3 UTRmRNA
Start Stop
NC
Nucleus
Cytoplasm2 315 UTR 4 3 UTR
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FORMATION OF AMINO ACYL-tRNA
- AMINO ACYL-tRNA SYNTHETASES :
* ENZYMES THAT ACTIVATE AMINO ACID
ATTACH THEM TO THE 3 TERMINAL ADENOSINE* PROOFREADING CAPABILITY
- HYDRALYZE THE AMINO ACID FROM THE tRNA
- THE AMYNO ACYLATION REACTION
AMINO ACID + ATP + tRNA AMINO ACYL- tRNA +P i + AMP
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Removal of introns from pre-tRNA.
The intron, located at the 3 end
of the antocordon, is removed.
Ligation produces the mature
tRNA.
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Nuclear ribonucleoproteins (snRNPs U1 to U6) bind to the intron causing it toform a loop.
Cleavage at the left splice sites releases exon 1, which remains attached tothe complex (the sliceosome). A 5-2 bond forms between the indicated Gand A residues.
A second cleavage releases exon 2, which is spliced to exon 1. The lariat-shaped intron is released.
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The mature, intronless mRNA, capped at the 5-end and equiped with a poly (A) tail,
complexes with proteins and travels through pores in the nuclear envelope into the
cytoplasm.
Overview of mRNA synthesis.
Transcription produces hnRNA from the DNA template.
hnRNA processing involves addition of a 5-cap and a poly (A) tail and splicing to join
exon and remove introns.
The product, mRNA, migrates to the cytoplasm.
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2 315 UTR 4 3 UTRmRNA
Start Stop
CCGGAUGCACUUGAAAUAAGCUA
GUG
H
UUU
K
CCGGAUGCACUUGAAAUAAGCUA
L
AAC
UAC
M
UAC
M
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CCGGAUGCACUUGAAAUAAGCUA
UACGUGAAC
GUG
MHL
K
GUG UAC
M
GUG
H
AAC
LK
CCGGAUGCACUUGAAAUAAGCUAGUG
H
UUU
K
CCGGAUGCACUUGAAAUAAGCUA
L
AAC
UAC
M
UAC
M
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Bonecell
Livercell
Neuron
Cell-typeCell-typespecificspecific
expressionexpression
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Cell-type specific expression
Temporal regulation
Coexpression with other genes
The transcription translation system allows
the formation of a complex life form such asa human being out of 30.000 genes
If everything goes fine
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Mutations
Abberant expression
Genetic disease
If something goes wrong
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Size of DNA molecules
- Human = 3.0 x 109 base pairs / haploid cell
= 6.0 x 109 base pairs / diploid cell- 1 chromosome ~ 7 cm in length- 46 chromosome 2 m- < 10% of DNA codes for a product
Packing of DNA in cells- DNA interacts with basic proteins known as histones
- Histones arginine and lysine- Five classes of histones : H1, H2A, H2B, H3 and H4
H2A
H2B EllipsoidH3
H4
- Other types of proteins are also associated with DNA
- Nucleosomes : * bead-like structures of DNA
* DNA coils around the surface of the ellipsoid
* Complex of histones plus DNA
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Multiple DNA polymerases with multiple enzymatic activities
(1). DNA polymerase I (pol I)
a. Function. Pol I functions in the replication of DNA and in the repair ofdamaged DNA
b. Other enzymatic activities. Pol I has two enzymatic activities -
besides DNA polymerase activity (1) Proofreading (Figure 1)
Figure 1. 3 to 5 Exonuclease (proofreading) activity
(2) Excision-repair (Figure 2). Pol I has a 5 to 3 exonuclease activity,
called excision-repair activity, that can hydrolytically remove a
segment of DNA from the 5 end of a strand of duplex DNA
(a) One to ten nucleotide segments of DNA can be removedat onetime
3 to 5
5 - G T C A T G G 5 - G T C A T G G .. .. .. ... .. .. .. ...3 - C A G T A C C G C T T A G - 5 3 - C A G T A C C G C T T A G - 5 + dTMP
exonuclease
3
-T-OH
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Figure 2. 5 to 3 exonuclease (excision-repair) activity.
3 to 5
G A A T G- 3 5 - T C -3 .. .. .. .. 3 - C A G T A C C G C T T A G - 5 3 - C A G T A C C G C T T A G - 5
+5 - T G A A - 3
exonuclease5-T-
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- Unwinds the supercoil DNA by cutting bothstrands of DNA
- Followed by the helicase activity
6. Single-strand binding protein ( SSBP )
it binds to single-stranded DNA (ssDNA)Function : - to protect ssDNA from nucleases
7. DNA Polymerases
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* Helicases unwind the parental DNA
* Single-strand binding proteins ( SSB )
prevent from reassociating
protection
- Action of DNA Polymerase
E.Coli has three DNA polymerase
Pol I
Pol IIPol III
- DNA gyrase uncoils DNA
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1. Unwind the helix by helicases
- With the aid of the DNA gyrase
- Dna B protein
- SSBP
2. DNA is replicated by DNA polymerase III.
The detail of the process :
- Primase ( a special RNA) a short RNA primer (10
nucleotides and compelemtary to the DNA)
- DNA polymerase III synthesizes complementary DNA
beginning at the 3 end of the RNA primer
- Leading and lagging strand are performed
- Lagging strand replcation is discontinous and the
fragments is called Okazaki fragments
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2. DNA polymerase also is essential for replication. It is
required for both leading and lagging strand synthesis
a. Pol associated with proliferating cell nuclear antigen(PCNA)
b. After pol has initiated replication, a protein calledreplication
factor C (RF-C) facilitates the inhibition an replacement
of pol with both PCNA and pol in an ATP-
dependent manner.
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3. DNA polymerase plays no role in replication and acts
only in DNA repair synthesis
4. DNA polymerase also is essential for replication,
although its exact role is not clearly defined. It behavessimilarly to pol and may support some component oflagging strand synthesis.
5. DNA polymerase replicates mitochondrial DNA
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1. 5 to 3 exonucleases. One is known to be associated with
DNA polymerase .
2. 3 to 5 exonucleases activity. DNA polymerase , , and have proofreading capability.
3. Ligase. Unlike prokaryotic ligase that use nicotinamideadenine dinucleotide (NAD) as an energy source,
eukaryotic ligases use
ATP
4. Helicase
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5. An SSBP called replication protein A (RP-A) has beenisolated from mammalian cells
6. Topoisomerases. These enzymes build up in advance
of replication forks.
Two basic types of eukaryotic topoisomerases.
a. Topoisomerase I is the major topoisomerase used to
relieve supercoils
b. Topoisomerase II also is required during replication.
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Model of the mechanism of replication of human telomeras
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MUTATIONS
Mutations permanent changes in the DNA
sequence.
Causes :
1. Errors in replication. If a base that noncomplementry tothe template base is added during replication, then the
error is not repaired mutation
2. Errors that accur during recombination events.
The DNA of living cells is surprisingly mobile and often isrearranged or recombined.
3. Chemical mutagens
4 Irradition
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4. Irradition
a. Ultraviolet ( UV ) light (200-400 nm) induces
dimerization of adjacent pyrimidines, particularly
adjacent thymines.B. Ionizing radiation, such as roentgen rays (x-rays) and
gamma rays ( -rays)5. Spontaneous changes. They lead to mutations if they
are not repaired before a round of replication.
a. Deamination of cytosine (C) to form uracil (U) occurs
spon-
taneously.
b. Spontaneous depurination. Purines are less stable
under normalcellular conditions than pyrimidines. The glycosidic
bond that
links purines to the sugar-phosphate backbone of
DNA oftenis broken
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1. Base substitution
- Base substitution are the most common type of
mutation, and they can be classified into twosubtypes :
(1). Transitions, in which one purine is replaced by
another purine or one pyrimidine is replaced by
another pyrimmidine
(2). Transversions, in which a purine is replaced by a
pyrimidiner a pyrimidine is replaced by a purine
2. Deletion of one or more base pairs
3. Insertion of one or more base pairs. Insertions of base
pairs intogenes can lead to severe frameshift mutation
(e.g., thalassemias)
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There are 2 types of changes:1. Point mutation gene mutation
mutation
2. Gross mutation chromosomeaberration aberration
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Abnormal structure or number ofchromosomes; includes deficiency,duplication, inversion, translocation,aneuploid, polyploid, or any changefrom normal
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A mutation caused by the altera- tion of asingle base in DNA:
Substitution of one base-pair for another
Duplication of single base-pairs
Deletion of single base-pairs
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a. MUTATION ACCORDING TO CHANGELOCATION
1. Mutation on nucleotides: deletion,
substitution, insertion, anddimerization
2. Mutation on splicing
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b. MUTATION IN ACCORDANCE WITH THEFORM OF CHANGES
1. Mis-sense mutation2. Non-sense mutation
3. Silence mutation
4. Frame-shift mutation
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1 Codon Deletion1 Codon Deletion
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1 Codon Deletion1 Codon DeletionCodogenCodogen ::TTATTATAGTAGTATAGG GAGAAA CCACCA CAACAA
CodonCodon :: AAU AUCAAU AUC AUCAUC CUUCUU GGUGGU GUUGUU
Amino acid: asn - ile - ile - leu - gly - valAmino acid: asn - ile - ile - leu - gly - val
Mutation on base sequence GGAMutation on base sequence GGA
CodogenCodogen ::TTATTATAGTAGTATACodonCodon :: AAU AUCAAU AUCAUAU
Amino acid: asn - ile -Amino acid: asn - ile -
AA CCACCA CAACAAUU GGUGGUGUUGUU
ile - gly - valile - gly - val
Frame-shift are occurred here, and proteiFrame-shift are occurred here, and proteiformed are distinctly different from theformed are distinctly different from the
originaloriginal
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1. A single-base substitution
Original
codon
Original
am. acid
New
codon
New
am. acid
UUA
GAU
Leu
Asp
UUG
GAA
Leu
Glu
SILENT MUTATION
MIS-SENSE MUTATION