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NUCLEIC ACID
Aulanni’am & indra Wibowo
Biochemistry Laboratory
Chemistry Departement
Brawijaya University
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NUCLEIC ACID
DNA, RNA, and Flow of Genetic Information
DNA (deoxyribonucleic acid)
RNA (ribonucleic acid)
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Central Dogma Biology Molecular/
Genetic Information
Replication
DNA
Transcription
Reverse Transcription
RNA
Translation
PROTEIN
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Genetic Information
ATGGTTTTCAGTGGAGTCATCCTTTCTGCTCTG
GTTATGTTTCTGCTTTCTGACAGTGCGCAGTG
CAGAAGAGTCGACTGCAAGACTGACTGTTGCT
CATTTGTGGAGGGCTTTCCAGTGAGACTCAAG
GAGCTCCGTTCTGCATACAGAGAAATACAGAA
CTTTTATGAGTCCAATGATGACATGGAACCATT
ACTGGACGAAAACGTGGAACAGAATATCAATA
GENETIC CODES
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Nucleic Acid Structure
base
base
sugar
base
sugar
phosphate
sugar
phosphate
base
base
sugar
sugar
phosphate
phosphate
phosphate
Structure of Nucleic Acids:
Primary structures both are linear polymers (multiple chemical units)
composed of monomers (single chemical units), called nucleotides
Functions of Nucleic Acids:
• contain the information prescribing amino acid sequence in proteins
• serve in the several cellular structures that choose, and then link into
the correct order, the amino acids of a protein chain
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Nucleotides are the Monomeric Units of Nucleic Acid
nucleoside
nucleotide
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First Component
RNA and DNA Differ in the Sugar Component
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Second Component
Phosphates
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Phosphodiester Linkage Formation
The chain-elongation reaction catalyzed by DNA polymerases is
a
nucleophilic attack by the 3’-hydroxyl group of the primer on the innermost
phosphorus atom of the deoxynucleoside triphosphate
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Backbones of DNA and RNA
3’ linkage
5’ linkage
Phosphodiester bond
RNA: 3’ 5’ phosphodiester bond
2’ 5’ phosphodiesterAulani
bond
(function
in RNA
Splicing)
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Function of the Nucleic Acid Backbones
negative charge
resistance to hydrolysis
To maintain the integrity of information
stored in nucleic acids
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Third Component
Purines and Pyrimidines
RNA
DNA
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Four Bases as Base Pairs of DNA
Specific hydrogen bonding between
G and C and between A and T
(or A and U) generates
complementary base-pairing
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β-Glycosidic Linkage in a Nucleoside
5’
4’
3’
1’
2’
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Naming Nucleosides and Nucleotides (Nomenclature)
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Naming Nucleosides and Nucleotides (Nomenclature)
Adenosine 5’-triphosphate (5’-ATP)/
5’-deoxyadenylate
Deoxyguanosine 3’-monophosphate (3’-dGMP)
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Structure of a DNA Chain
• A DNA chain has polarity.
• One end has a free 5’-OH group attach
to a phosphate
• Other end has a 3’-OH group
• The base sequence is written in
the 5’ to 3’ direction
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A Pair of Nucleic Acid Chains with Complementary
Sequences Can Form a Double-Helical Structure
X-Ray Diffraction Photograph of
a Hydrated DNA Fiber
(Maurice Wilkins and Rosalind Franklin)
Watson-Crick Model of Double-Helical DNA
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Watson-Crick Model of Double Stranded-DNA
34 Å
• Helix
• Antiparallel, hydrogen bond
• Sugar-phosphate backbones outside, bases inside the helix, minor and major grooves
• Bases and axis nearly perpedicular
• Helix diameter 2 nm (20 Å)
• Adjacent bases are separated by 3.4 Å
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• The helical structure repeats every 34 Å (10 bases/turn)
The Double Helix is Stabilized by Hydrogen Bonds
and Hydrophobic Interactions
The stacking of bases one on top of
another contributes to the stability
of the double helix in two ways:
1. van der Waals interactions
2. hydrophobic effect
Rigid five-carbon sugar (pentose)
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Two Possible Helical Forms of DNA are
Mirror Images of Each Other
The geometry of the sugar-phosphate backbone of DNA causes
natural DNA to be right-handed
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Models of Various DNA Structures
that are Known to Exist
• The B form of DNA, the usual
form in cells, is characterized by
a helical turn every 10 base pairs
(3.4 nm)
• The more compact A form of
DNA has 11 base pairs per turn
and exhibits a large tilt of the
base pairs with respect to the
helix axis
• Z DNA is a left-handed helix
and has a zig-zag (hence "Z")
appearance
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Some DNA Molecules are Circular and Supercoiled
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The Denaturation and Renaturation of
Double-Stranded DNA Molecules
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The Double Helix Facilitates the Accurate
Transmission of Hereditary Information
DNA Synthesis is catalyzed by DNA Polymerases
Occur at all places of DNA chain,
5’3’ direction
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Semiconservative
RNA Molecules Exhibit Varied
Conformations and Functions
Several Kinds of RNA Play Key Roles in Gene Expression
• mRNA (messenger RNA): is the template for protein synthesis or translation
• tRNA (transfer RNA): carries amino acids in an activated form to the ribosome for peptide-
bond formation
• rRNA (ribosomal RNA): the major component of ribosomes
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Structural Comparisons between DNA and RNA
RNA
DNA
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Central Dogma Biology Molecular/
Genetic Information
DNA
Replication
Transcription
Reverse Transcription
RNA
Translation
PROTEIN
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Transcription
mRNA
-strand
Template strand of DNA (antisense)
Coding strand of DNA (sense)
+strand
• Transcription Mechanism of the Chain-Elongation Reaction
Catalyzed by RNA Polymerase
• 5’3’ direction
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Promoter Sites for Transcription
Start signals are required for the initiation of RNA synthesis in
(A) prokaryotes and (B) eukaryotes
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Transcription, Translation and Reverse Transcription
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The Genetic Code
1. Three nucleotides encode an amino acid
2. The code is nonoverlapping
3. The code has no punctuation
4. The genetic code is degenerate
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The Genetic Code
Codon: A three-nucleotide sequence of DNA or mRNA that specifies a particular
amino acid or termination signal; the basic unit of the genetic code
Anticodon: A specialized base triplet at one end of a tRNA molecule that
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recognizes a particular
complementary
on an mRNA molecule
tRNA and rNA
Phenylalanine tRNA of yeast
The structure of the rRNA in the small subunit
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RNA Processing Generates Mature RNA
intron
exon
Splicing
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Translation
codon
Synthesis of a protein by ribosomes
attached to an mRNA molecule.
anticodon
Translation of the mRNA nucleotide
sequence into an amino acid sequence
depends on complementary base-pairing
between codons in the mRNA and
corresponding tRNA anticodons.
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Recombinant DNA Technology
• Fragmentation, Separation, and
Sequencing of DNA Molecules
• DNA Cloning
•DNA Engineering
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Recombinant DNA Technology
(Palindrome, Restriction Enzyme, Sticky Ends)
Sticky Ends
(Cohesive Ends)
G
AATTC
AATTC
GAATTC
EcoRI
GAATTC
Blunt End
TTC
G AATTC
G AATTC
GAA
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G
CIVIC, Madam
TTC
GAA
Recombinant DNA Technology
(Restriction Mapping)
Restriction
enzymes
U A
-
B A+B M
A
A 8 kb
2 kb
B 7 kb
3 kb
+
A 5 kb
+ 3 kb
B 2 kb
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B
10 kb
Recombinant DNA Technology
(Restriction and Ligation)
GAATTC
GAATTC
CTTAAG
CTTAAG
EcoRI
G AATTC
G AATTC
CTTAA
CTTAA
G
G
G AATTC
CTTAA
CTTAA
EcoRI sticky end
G
DNA Ligase
G AATTC
CTTAA G
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G
AATTC
G
EcoRI sticky end
Recombinant DNA Technology
(Random Fragment Length Polymorfism)
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Recombinant DNA Technology
(Random Fragment Length Polymorfism)
recombination
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Recombinant DNA Technology
(Sequencing)
Sanger Method:
ddNTP
H
H
Dideoxyadenosine 5’-triphosphate (ddNTP)
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Recombinant DNA Technology
(DNA Cloning: Drug Resistance Gene Transferred by Plasmid )
Drug Resistant Gene
mRNA
Plasmid
Resistant Strain
Plasmid gets out and
into the host cell
Enzyme
Hydrolyzing
Antibiotics
New Resistance Strain
Non-resistant Strain
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Target Genes
Restriction
Enzyme
Restriction
Enzyme
DNA Recombination
Target Gene
Recombination
Transformation
Host Cells
Recombinant
Plasmid
Transformation
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Chromosomal DNA
Recombinant DNA Technology
(DNA Cloning: Target Genes Carried by Plasmid)
1 plasmid
1 cell
Recombinant DNA Technology
(DNA Cloning: Amplification and Screening of Target Gene)
1
Plating
1 cell line, 1 colony
Bacteria
Duplication
X1,000
Pick the colony
containing target gene
=100,000
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X100
Plasmid
Duplication
Recombinant DNA Technology
(Libraries: Intron and Exon Organization)
exon
promotor
3’
exon
intron
exon
intron
DNA
5’
stop codon
start codon
Transcription
5’
3’ mRNA
Processing
cap
poly A
tail
Splicing
Intron deleted
Take place in nucleus
mature mRNA
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To cytoplasm
Recombinant DNA Technology
(Libraries: cDNA Synthesis)
5’
Reverse
transcription
3’
mature mRNA
RNA
hydrolysis
3’
3’
poly A tail
TTTT
5’
5’
DNA
polymerase
3’ CCC
5’ GGG
5’
3’
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Recombinant DNA Technology
(Libraries: cDNA and Genomic)
Genes in expression
Total Gene
Chromosomal DNA
mRNA
Reverse transcription
cDNA
Vector: Plasmid
Restriction digestion
Complete
gene
Gene fragments
Smaller
Library
Larger
Library
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Vector:
Plasmid or phage
Recombinant DNA Technology
(DNA Engineering: Polymerase Chain Reaction)
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Recombinant DNA Technology
(PCR for Forensic Science or PRC Fingerprint)
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Recombinant DNA Technology
(Antisense RNA Strategy)
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Recombinant DNA Technology
(Gene Knockout)
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