aulani "biokimia" presentation 9 nucleic acid aulanni’am & indra wibowo biochemistry...

Aulani "Biokimia" Presentation 9

NUCLEIC ACID

Aulanni’am & indra WibowoBiochemistry LaboratoryChemistry Departement

Brawijaya University


NUCLEIC ACID

DNA, RNA, and Flow of Genetic Information

DNA (deoxyribonucleic acid)RNA (ribonucleic acid)


DNA

RNA

PROTEIN

Transcription

Translation

Reverse Transcription

Replication

Central Dogma Biology Molecular/Genetic Information


Genetic Information

ATGGTTTTCAGTGGAGTCATCCTTTCTGCTCTGGTTATGTTTCTGCTTTCTGACAGTGCGCAGTGCAGAAGAGTCGACTGCAAGACTGACTGTTGCTCATTTGTGGAGGGCTTTCCAGTGAGACTCAAGGAGCTCCGTTCTGCATACAGAGAAATACAGAACTTTTATGAGTCCAATGATGACATGGAACCATTACTGGACGAAAACGTGGAACAGAATATCAATA

GENETIC CODES


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


Nucleotides are the Monomeric Units of Nucleic Acid

nucleoside

nucleotide


RNA and DNA Differ in the Sugar ComponentFirst Component


PhosphatesSecond Component


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


Backbones of DNA and RNA

Phosphodiester bond

3’ linkage 5’ linkage

RNA: 3’ 5’ phosphodiester bond 2’ 5’ phosphodiester bond (function in RNA Splicing)


To maintain the integrity of information stored in nucleic acids

negative charge

resistance to hydrolysis

Function of the Nucleic Acid Backbones


Purines and Pyrimidines

RNA DNA

Third Component


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


β-Glycosidic Linkage in a Nucleoside

1’2’3’

4’

5’


Naming Nucleosides and Nucleotides (Nomenclature)


Adenosine 5’-triphosphate (5’-ATP)/5’-deoxyadenylate

Deoxyguanosine 3’-monophosphate (3’-dGMP)

Naming Nucleosides and Nucleotides (Nomenclature)


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


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


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


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)


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


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


Some DNA Molecules are Circular and Supercoiled


The Denaturation and Renaturation of Double-Stranded DNA Molecules


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


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


Structural Comparisons between DNA and RNA

DNA

RNA


DNA

RNA

PROTEIN

Transcription

Translation

Reverse Transcription

Replication

Central Dogma Biology Molecular/Genetic Information


• 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


Promoter Sites for Transcription

Start signals are required for the initiation of RNA synthesis in(A) prokaryotes and (B) eukaryotes


Transcription, Translation and Reverse Transcription


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


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


tRNA and rNA

The structure of the rRNA in the small subunit

Phenylalanine tRNA of yeast


RNA Processing Generates Mature RNA

Splicing

exon

intron


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


Recombinant DNA Technology

• Fragmentation, Separation, and Sequencing of DNA Molecules

• DNA Cloning

•DNA Engineering


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


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)

-

+


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)


Recombinant DNA Technology(Random Fragment Length Polymorfism)


Recombinant DNA Technology(Random Fragment Length Polymorfism)

recombination


Recombinant DNA Technology (Sequencing)

Sanger Method: ddNTP

Dideoxyadenosine 5’-triphosphate (ddNTP)

H H


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 )


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)


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)


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)


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)


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)


Recombinant DNA Technology(DNA Engineering: Polymerase Chain Reaction)


Recombinant DNA Technology(PCR for Forensic Science or PRC Fingerprint)


Recombinant DNA Technology(Antisense RNA Strategy)


Recombinant DNA Technology(Gene Knockout)

aulani "biokimia" presentation 9 nucleic acid aulanni’am & indra wibowo biochemistry...

Documents

aulani biokimia presentation

component slide

direction slide

nucleic acid dna

base pairs of dna slide

rna splicing slide

nucleotides nomenclature

protein chain slide