28-1 nucleic acids. 28-2 nucleic acids components nucleic acid: nucleic acid: a biopolymer...
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Nucleic AcidsNucleic Acids
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Nucleic Acids ComponentsNucleic Acids Components
Nucleic acid:Nucleic acid: A biopolymer containing three types of monomer units.• Heterocyclic aromatic amine bases derived from
purine and pyrimidine.• The monosaccharides D-ribose or 2-deoxy-D-ribose• Phosphoric acid.
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Purine/Pyrimidine BasesPurine/Pyrimidine Bases
HN
N
O
O
H
N
N
NH2
O
H
HN
N
O
O
H
CH3N
N
HN
N N
N
O
HH2N
N
N N
N
NH2
H
N
N N
N
H
Uracil (U) Thymine (T) Cytosine (C)Pyrimidine
1
2
34
5
6
Guanine (G)Adenine (A)Purine
1
2
34
56 7
8
9
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NucleoNucleossides Definitionides Definition
Nucleoside:Nucleoside: A building block consisting of • D-ribose or 2-deoxy-D-ribose • heterocyclic aromatic amine base • -N-glycosidic bond .
HH
HOHOCH2
HO OH
O
O
HN
N
H anomericcarbon
a -N-glycosidicbond
Uridine
-D-riboside
uracil
1'
2'3'
4'
5'
1
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NucleoNucleottides Definitionides Definition
NucleoNucleottide:ide: Phosphoric acid ester of a nucleoside, most commonly either the 3’ or the 5’ OH.
N
NN
N
NH2
O
OHOH
HH
H
CH2
H
OP
O-
O-O
5'
Adenosine 5'-monophosphate(AMP)
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Acyclovir & AZTAcyclovir & AZT
Azidothymidine (AZT) Acyclovir (drawn to show itsstructural relationship to 2-deoxyguanosine
O
O
N
HN
HOC
HN3
H
H
HH
O
CH3
O
H HHH
HOC
H2N
HN
N
O
N
N
H2H2
Used to treat HIV
It is used to treat or prevent infections caused by certain kinds of viruses. Examples of these infections include herpes and shingles
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DNA - 1° StructureDNA - 1° Structure
Deoxyribonucleic acids (DNA)• A backbone of alternating units of 2-deoxy-D-ribose
and phosphate in which the 3’-OH of one 2-deoxy-D-ribose is joined by a phosphodiester bond to the 5’-OH of another 2-deoxy-D-ribose.
Primary Structure:Primary Structure: The sequence of bases along the pentose-phosphodiester backbone of a DNA molecule (or an RNA molecule). • Read from the 5’ end to the 3’ end.
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DNA - 1° StructureDNA - 1° Structure
• A structural formula for TG phosphorylated at the 5’ end.
NO
HN
-O-P-O-CH2
O
HH
OH
HH
H
OCH3
O=P O CH2
O
-O
O-
NH
NN
N
O
NH2
O
HH
O
HH
H
5'
3'
phosphorylated5' end
free 3' end
Thymine, T
Guanine, G3
diester
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DNA - 2° StructureDNA - 2° Structure
Secondary structure:Secondary structure: The ordered arrangement of nucleic acid strands.
The double helix model of DNA 2° structure was proposed by James Watson and Francis Crick in 1953.
Double helix:Double helix: A type of 2° structure of DNA molecules in which two antiparallel polynucleotide strands are coiled in a right-handed manner about the same axis.
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DNA - 2° StructureDNA - 2° Structure
• Hydrogen bonding occurs between bases• A---T
• G---C
• Evidence: Base composition in mole-percent of DNA for several organisms.
A C TOrganism G A/T G/CPurines/Pyrimidines
HumanSheepYeast
E. coli
30.4 19.9 19.9 30.1 1.01 1.00 1.0129.3 21.4 21.0 28.3 1.04 1.02 1.0331.7 18.3 17.4 32.6 0.97 1.05 1.0026.0 24.9 25.2 23.9 1.09 0.99 1.04
Purines Pyrimidines
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Base PairingBase Pairing
• Base-pairing between adenine and thymine (A-T) and guanine and cytosine (G-C).
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Double HelixDouble Helix
• Ribbon model of double-stranded B-DNA.
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Forms of DNAForms of DNA
B-DNA • the predominant form in dilute aqueous solution.• a right-handed helix.• 2000 pm thick with 3400 pm per ten base pairs.• minor groove of 1200pm and major groove of 2200 pm.
A-DNA• a right-handed helix, but thicker than B-DNA.• 2900 pm per 10 base pairs.
Z-DNA• a left-handed double helix.
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Double HelixDouble Helix
• An idealized model of B-DNA.
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DNA - 3° StructureDNA - 3° Structure
Tertiary structure:Tertiary structure: The three-dimensional arrangement of all atoms of a double-stranded DNA, commonly referred as supercoiling.
Circular DNA:Circular DNA: A type of double-stranded DNA in which the 5’ and 3’ ends of each stand are joined by phosphodiester bonds.
Histone:Histone: A protein, particularly rich in the basic amino acids lysine and arginine, that is found associated with DNA molecules.
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DNA - 3° StructureDNA - 3° Structure
Chromatin:Chromatin: Consists of DNA molecules wound around particles of histones (a simple protein containing mainly basic amino acids;) in a beadlike structure.• Further coiling produces the dense chromatin found in
nuclei of plant and animal cells.
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Ribonucleic Acids (RNA)Ribonucleic Acids (RNA)
RNA • long, unbranched chains of nucleotides joined by
phosphodiester groups between the 3’-OH of one pentose and the 5’-OH of the next;
• Consists of A, U ( Uracil), G, C.• the pentose unit in RNA is -D-ribose rather than -2-
deoxy-D-ribose.• the pyrimidine bases in RNA are uracil and cytosine
rather than thymine and cytosine.• RNA is single stranded rather than double stranded.
(RNA)
A –Uracil
C –Cytosine
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rRNArRNA
Different types of RNA:
Ribosomal RNA (rRNA):Ribosomal RNA (rRNA): A ribonucleic acid found in ribosomes, the site of protein synthesis.
Molecular WeightRange (g/mol)
Number ofNucleotides
Percentageof Cell RNA
mRNA 25,000 - 1,000,000 75 - 3,000 2tRNA 23,000 - 30,000 73 - 94 16rRNA 35,000 - 1,100,000 120 - 2904 82
Type
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tRNAtRNA
Transfer RNA (tRNA):Transfer RNA (tRNA): A ribonucleic acid that carries a specific amino acid to the site of protein synthesis on ribosomes.
OBase
OHO
CH
RNH3
+
tRNA-O-P-O-CH2
amino acid, boundas an ester to itsspecific tRNA
HH H
H
C=O
O
O-
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mRNAmRNA
Messenger RNA (mRNA):Messenger RNA (mRNA): A ribonucleic acid that carries coded genetic information from DNA to the ribosomes for the synthesis of proteins.• Present in cells in relatively small amounts and very
short-lived.• Single stranded.• mRNA synthesis is directed by information encoded
on DNA.• A complementary strand of mRNA is synthesized
along one strand of an unwound DNA, starting from the 3’ end.
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mRNA from DNA, transcriptionmRNA from DNA, transcription
DNA (RNA)
A – T(U)
G – C(C)
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UUU PheUUC PheUUA LeuUUG Leu
UCU SerUCC SerUCA SerUCG Ser
UAU TyrUAC TyrUAA StopUAG Stop
UGU CysUGC CysUGA StopUGG Trp
GUCAG
CUU LeuCUC LeuCUA LeuCUG Leu
CCU ProCCC ProCCA ProCCG Pro
CAU HisCAC HisCAA GlnCAG Gln
CGU ArgCGC ArgCGA ArgCGG Arg
UCAG
G
AUU IleAUC IleAUA IleAUG* Met
ACU ThrACC ThrACA ThrACG Thr
AAU AsnAAC AsnAAA LysAAG Lys
AGU SerAGC SerAGA ArgAGG Arg
UCAG
UCAG
GUU ValGUC ValGUA ValGUG Val
GCU AlaGCC AlaGCA AlaGCG Ala
GAU AspGAC AspGAA GluGAG Glu
GGU GlyGGC GlyGGA GlyGGG Gly
*AUG also serves as the principal initiation codon.
U
U C A
C
A
The Genetic CodeThe Genetic Code
first
second
third
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The Genetic CodeThe Genetic Code
Properties of the Code• Only 61 triplets code for amino acids; the remaining 3
(UAA, UAG, and UGA) signal chain termination.• The code is degenerate, which means that several
amino acids are coded for by more than one triplet. Leu, Ser, and Arg, for example, are each coded for by six triplets.
• Degenerate triplets differ only in the third letter of the codon that varies. Gly, for example, is coded for by GGA, GGG, GGC, and GGU. (GG? Codes for GLY)
• There is no ambiguity in the code; each triplet codes for one and only one amino acid.
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Sequencing DNASequencing DNA
Restriction endonuclease:Restriction endonuclease: An enzyme that catalyzes hydrolysis of a particular phosphodiester bond within a DNA strand.• Over 1000 endonucleases have been isolated and their
specificities determined.• Typically they recognize a set sequence of nucleotides
and cleave the DNA at or near that particular sequence.
• EcoRI (eco R 1) from E. coli, for example, cleaves as shown.
5' G-A-A-T-T-C---3'EcoRI
5' G + 5'-A-A-T-T-C---3'
cleavage here
Recognition pattern
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Sequencing DNASequencing DNA
• Following are several more examples of endonucleases and their specificities.
AluI AG CT
BalI TGG CCA
FnuDII CG CG
HeaIII GG CC
Not I GC GGCCGC
Mbol GATC
SacI GAGCT C
Enzyme EnzymeRecognitionSequence
RecognitionSequence
HpaII C CGG
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Sequencing DNASequencing DNA
• Maxam-Gilbert method:Maxam-Gilbert method: A method developed by Allan Maxam and Walter Gilbert; depends on base-specific chemical cleavage.
• Dideoxy chain termination methodDideoxy chain termination method:: Developed by Frederick Sanger.
Gilbert and Sanger shared the 1980 Nobel Prize for biochemistry for their “development of chemical and biochemical analysis of DNA structure.”
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ReplicationReplication in Vitro in Vitro
• the sequence of nucleotides in one strand (ssDNA) is copied as a complementary strand to form the second strand of a double-stranded DNA (dsDNA).
• Synthesis is catalyzed by DNA polymerase.• DNA polymerase requires
• the four deoxynucleotide triphosphate (dNTP) monomers
• a primer is present to start the process.
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Dideoxy Chain TerminationDideoxy Chain Termination
• Chain termination method is accomplished by the addition to the synthesizing medium of a 2’,3’-dideoxynucleotide triphosphate (ddNTP).
• Because a ddNTP has no 3’-OH, chain synthesis is terminated when a ddNTP becomes incorporated.
-O-P-O-P-O-P-O-CH2
O-
O
O- O-
O
H
Base
H H
H HO
H
A 2',3'-dideoxynucleoside triphosphate (ddNTP)
O
Without a OH here chain cannot extend.
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MethodologyMethodology of Dideoxy Chain Termination of Dideoxy Chain Termination
In this method, the following are mixed:• Single-stranded DNA of unknown sequence and
primer; then divided into four reaction mixtures.
To each of the four reaction mixture is then added:• The four dNTP, one of which is labeled in the 5’ end
with phosphorus-32 which is radioactive.• DNA polymerase.
• one of the four ddNTPs.
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Dideoxy Chain TerminationDideoxy Chain Termination
After gel electrophoresis of each reaction mixture• a piece of film is placed over the gel.• Gamma rays released by P-32 darken the film and
create a pattern of the resolved oligonucleotide.• The base sequence of the complement to the original
strand is read directly from bottom to top of the developed film.
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Dideoxy Chain TerminationDideoxy Chain Termination
• The primer-DNA template is divided into four separate reaction mixtures. To each is added the four dNTPs, DNA polymerase, primer and one of the four ddNTPs in small amounts. Synthesis
will produce chains of varying lengths.
DNA
A – T
G – C
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Dideoxy Chain TerminationDideoxy Chain Termination
The mixtures are separated by polyacrylamide gel electrophoresis. From the four different ddNTP reaction mixtures.
Move from 5’ to 3’ end.