Post on 18-Dec-2015
Aulani "Biokimia" Presentation 9
NUCLEIC ACID
Aulanni’am & indra WibowoBiochemistry LaboratoryChemistry Departement
Brawijaya University
Aulani "Biokimia" Presentation 9
NUCLEIC ACID
DNA, RNA, and Flow of Genetic Information
DNA (deoxyribonucleic acid)RNA (ribonucleic acid)
Aulani "Biokimia" Presentation 9
DNA
RNA
PROTEIN
Transcription
Translation
Reverse Transcription
Replication
Central Dogma Biology Molecular/Genetic Information
Aulani "Biokimia" Presentation 9
Genetic Information
ATGGTTTTCAGTGGAGTCATCCTTTCTGCTCTGGTTATGTTTCTGCTTTCTGACAGTGCGCAGTGCAGAAGAGTCGACTGCAAGACTGACTGTTGCTCATTTGTGGAGGGCTTTCCAGTGAGACTCAAGGAGCTCCGTTCTGCATACAGAGAAATACAGAACTTTTATGAGTCCAATGATGACATGGAACCATTACTGGACGAAAACGTGGAACAGAATATCAATA
GENETIC CODES
Aulani "Biokimia" Presentation 9
phosphate
sugar
base
phosphate
sugar
base
phosphate
sugar
base
Structure of Nucleic Acids:
Primary structures both are linear polymers (multiple chemical units)
composed of monomers (single chemical units), called nucleotides
sugar
phosphate
base
sugar
phosphate
base
Nucleic Acid Structure
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
Aulani "Biokimia" Presentation 9
Nucleotides are the Monomeric Units of Nucleic Acid
nucleoside
nucleotide
Aulani "Biokimia" Presentation 9
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
Aulani "Biokimia" Presentation 9
Backbones of DNA and RNA
Phosphodiester bond
3’ linkage 5’ linkage
RNA: 3’ 5’ phosphodiester bond 2’ 5’ phosphodiester bond (function in RNA Splicing)
Aulani "Biokimia" Presentation 9
To maintain the integrity of information stored in nucleic acids
negative charge
resistance to hydrolysis
Function of the Nucleic Acid Backbones
Aulani "Biokimia" Presentation 9
Specific hydrogen bonding between G and C and between A and T (or A and U) generates complementary base-pairing
Four Bases as Base Pairs of DNA
Aulani "Biokimia" Presentation 9
Adenosine 5’-triphosphate (5’-ATP)/5’-deoxyadenylate
Deoxyguanosine 3’-monophosphate (3’-dGMP)
Naming Nucleosides and Nucleotides (Nomenclature)
Aulani "Biokimia" Presentation 9
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
Aulani "Biokimia" Presentation 9
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
Aulani "Biokimia" Presentation 9
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 • The helical structure repeats every 34 Å (10 bases/turn)
Watson-Crick Model of Double Stranded-DNA
Aulani "Biokimia" Presentation 9
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)
Aulani "Biokimia" Presentation 9
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
Aulani "Biokimia" Presentation 9
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
Aulani "Biokimia" Presentation 9
DNA Synthesis is catalyzed by DNA PolymerasesOccur at all places of DNA chain, 5’3’ direction Semiconservative
The Double Helix Facilitates the Accurate Transmission of Hereditary Information
Aulani "Biokimia" Presentation 9
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
RNA Molecules Exhibit Varied Conformations and Functions
Aulani "Biokimia" Presentation 9
DNA
RNA
PROTEIN
Transcription
Translation
Reverse Transcription
Replication
Central Dogma Biology Molecular/Genetic Information
Aulani "Biokimia" Presentation 9
• Transcription Mechanism of the Chain-Elongation Reaction Catalyzed by RNA Polymerase • 5’3’ direction
Transcription
mRNA
Template strand of DNA (antisense)
Coding strand of DNA (sense)
-strand
+strand
Aulani "Biokimia" Presentation 9
Promoter Sites for Transcription
Start signals are required for the initiation of RNA synthesis in(A) prokaryotes and (B) eukaryotes
Aulani "Biokimia" Presentation 9
1. Three nucleotides encode an amino
acid
2. The code is nonoverlapping
3. The code has no punctuation
4. The genetic code is degenerate
The Genetic Code
Aulani "Biokimia" Presentation 9
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 recognizes a particular complementary codon on an mRNA molecule
Aulani "Biokimia" Presentation 9
tRNA and rNA
The structure of the rRNA in the small subunit
Phenylalanine tRNA of yeast
Aulani "Biokimia" Presentation 9
Translation
Synthesis of a protein by ribosomes attached to an mRNA molecule.
Translation of the mRNA nucleotide sequence into an amino acid sequence depends on complementary base-pairingbetween codons in the mRNA and corresponding tRNA anticodons.
codon
anticodon
Aulani "Biokimia" Presentation 9
Recombinant DNA Technology
• Fragmentation, Separation, and Sequencing of DNA Molecules
• DNA Cloning
•DNA Engineering
Aulani "Biokimia" Presentation 9
GAATTCGAATTC
GAATTC G
AATTC
Sticky Ends(Cohesive Ends)
EcoRI
CIVIC, Madam
G
AATTC G
AATTC
Recombinant DNA Technology(Palindrome, Restriction Enzyme, Sticky Ends)
GAA
TTC GAA
TTCBlunt End
Aulani "Biokimia" Presentation 9
A B 10 kb
8 kb2 kb
A
7 kb3 kb
B
5 kb3 kb2 kb
A+B
U A B A+B M
Restriction enzymes
Recombinant DNA Technology (Restriction Mapping)
-
+
Aulani "Biokimia" Presentation 9
GAATTC
CTTAAG
GAATTC
CTTAAG
G
CTTAA
AATTC
G
AATTC
G
G
CTTAA
G
CTTAA
AATTC
G
G
CTTAA
AATTC
G
G
CTTAA
AATTC
G
EcoRI
DNA LigaseEcoRI sticky end EcoRI sticky end
Recombinant DNA Technology (Restriction and Ligation)
Aulani "Biokimia" Presentation 9
Recombinant DNA Technology(Random Fragment Length Polymorfism)
recombination
Aulani "Biokimia" Presentation 9
Recombinant DNA Technology (Sequencing)
Sanger Method: ddNTP
Dideoxyadenosine 5’-triphosphate (ddNTP)
H H
Aulani "Biokimia" Presentation 9
Plasmid gets out and into the host cell
Resistant Strain
New Resistance Strain
Non-resistant Strain
Plasmid
EnzymeHydrolyzingAntibiotics
Drug Resistant Gene
mRNA
Recombinant DNA Technology(DNA Cloning: Drug Resistance Gene Transferred by Plasmid )
Aulani "Biokimia" Presentation 9
1 plasmid1 cellRecombinant
PlasmidTransformation
Target GeneRecombination
Restriction
Enzyme
Restriction
Enzyme
Ch
rom
oso
mal
DN
ATarget Genes
DNA Recombination
TransformationHost Cells
Recombinant DNA Technology (DNA Cloning: Target Genes Carried by Plasmid)
Aulani "Biokimia" Presentation 9
1
1 cell line, 1 colony
X100
X1,000
PlasmidDuplicationBacteria
Duplication
Plating
Pick the colonycontaining target gene
=100,000
Recombinant DNA Technology(DNA Cloning: Amplification and Screening of Target Gene)
Aulani "Biokimia" Presentation 9
mRNA
DNA
5’ 3’
cap
poly Atail
exon exonexonintron intron
mature mRNA
Processing
Transcription
Splicing
promotor
3’ 5’
Take place in nucleus
start codon stop codon
To cytoplasm
Intron deleted
Recombinant DNA Technology(Libraries: Intron and Exon Organization)
Aulani "Biokimia" Presentation 9
mature mRNA
poly A tail5’ 3’
TTTTReverse transcription
CCC
3’ 5’
3’5’ 3’GGG
DNA polymerase
RNA hydrolysis
5’
3’ 5’
Recombinant DNA Technology (Libraries: cDNA Synthesis)
Aulani "Biokimia" Presentation 9
mRNA
cDNA
Reverse transcription
Chromosomal DNA
Restriction digestion
Genes in expression Total Gene
Complete gene Gene fragments
SmallerLibrary
Larger Library
Vector:Plasmid or phageVector: Plasmid
Recombinant DNA Technology (Libraries: cDNA and Genomic)
Aulani "Biokimia" Presentation 9
Recombinant DNA Technology(DNA Engineering: Polymerase Chain Reaction)
Aulani "Biokimia" Presentation 9
Recombinant DNA Technology(PCR for Forensic Science or PRC Fingerprint)