©2000 timothy g. standish structure and analysis of dna

©2000 Timothy G. Standish

Structure and AnalysisStructure and Analysisofof

DNA DNA

.

DNA

mRNA

Transcription

IntroductionIntroduction

The Central Dogma The Central Dogma of Molecular Biologyof Molecular Biology

Cell

Polypeptide(protein)

TranslationRibosome



OutlineOutline

1How we know DNA is the genetic material

2Basic structure of DNA and RNA

3Ways in which DNA can be studied and what they tell us about genomes


Historical EventsHistorical Events• 1869 Friedrich Miescher identified DNA, which he called nuclein,

from pus cells• 1889 Richard Altman renamed nuclein nucleic acid• 1928 Griffith discovered that genetic information could be passed

from one bacteria to another; known as the transforming principle• 1944 Avery showed that the transforming material was pure

DNA not protein, lipid or carbohydrate.• 1952 Hershey and Chase used bacteriophage (virus) and E. coli

to show that only viral DNA entered the host• 1953 Watson and Crick discovered the structure of DNA was a

double helix


Transformation Of BacteriaTransformation Of BacteriaTwo Strains Of Two Strains Of StreptococcusStreptococcus

Capsules

Smooth Strain(Virulent)

Rough Strain(Harmless)


Experimental

Transformation Of BacteriaTransformation Of BacteriaThe Griffith’s 1928 ExperimentThe Griffith’s 1928 Experiment

- Control

+ Control

- Control

OUCH!


Avery, MacLeod and McCartyAvery, MacLeod and McCarty 1944 Avery, MacLeod and McCarty repeated Griffith’s

1928 experiment with modifications designed to discover the “transforming factor”

After extraction with organic solvents to eliminate lipids, remaining extract from heat killed cells was digested with hydrolytic enzymes specific for different classes of macro molecules:

NoNuclease

YesProtease

Transformation?Enzyme

YesSaccharase


The Hershey-Chase The Hershey-Chase ExperiementExperiement

The Hershey-Chase experiment showed definitively that DNA is the genetic material

Hershey and Chase took advantage of the fact that T2 phage is made of only two classes of macromolecules: Protein and DNA

HOH

P

O

OH

HO ONH2

Nucleotides contain phosphorous, thus DNA contains phosphorous, but not sulfur.

H

OH

OH2N CC

CH2

SH

H

OH

OH2N C

CH3

C

CH2

CH2

S Some amino acids contain sulfur, thus proteins contain sulfur, but not phosphorous.

CysteineMethionine

Using SUsing S3535Bacteria grown in normal non-radioactive media

T2 grown in S35 containing media incorporate S35 into their proteins

Blending causes phage protein coat to fall off

T2 attach to bacteria and inject genetic material

Is protein the genetic material?

When centrifuged, phage protein coats remain in the supernatant while bacteria form a pelletThe supernatant is radioactive, but the pellet is not.

Did protein enter the bacteria?

Using PUsing P3232Bacteria grown in normal non-radioactive media

T2 grown in P32 containing media incorporate P32 into their DNA

Blending causes phage protein coat to fall off

T2 attach to bacteria and inject genetic material

Is DNA the genetic material?

When centrifuged, phage protein coats remain in the supernatant while bacteria form a pelletThe pellet is radioactive, but the supernatant is not.

Did DNA enter the bacteria?

OH

OCH2

Sugar

H

HH

A NucleotideA NucleotideAdenosine Mono Phosphate (AMP)Adenosine Mono Phosphate (AMP)

OH

NH2

N

N N

N

BaseP

O

OH

HO O

Phosphate

2’3’

4’

5’

1’Nucleotide

Nucleoside

H+

-

Pyrimidines

NH2

O

N

N NH

N

Guanine

N

N

Adenine

N

N

NH2

N O

NH2

N O

NH2

NCytosine

Purines

Uracil(RNA)CH3

N ON

O

NH

N ON

O

NH

Thymine(DNA)

NO

H

NO

N

NH C

ytosine

H

O

NN

N

N

N

H

H

Guanine -+

+

+

-

-

Base PairingBase PairingGuanine And CytosineGuanine And Cytosine

CH 3

N

O

N

ON

H+

- ThymineN

NN

N

HN H

-

+Adenine

Base PairingBase PairingAdenine And ThymineAdenine And Thymine

Base PairingBase PairingAdenine And CytosineAdenine And Cytosine

NO

H

NO

N

NH C

ytosine-

+

-

N

NN

N

HN

H

-

+

Adenine

Base PairingBase PairingGuanine And ThymineGuanine And Thymine

CH

3

NO

N

O

NH+

- Thymine

H

O

NN

N

N

N

H

H

Guanine

+

+

-


Some minor purine and Some minor purine and pyrimidine basespyrimidine bases

SU

GA

R-P

HO

SP

HA

TE

BA

CK

BO

NE

H

P

O

HO

O

O

CH2

HOH

P

O

O

HO

O

O

CH2

H

P

O

OH

HO

O

O

CH2

NH2

N

N

N

N

O

O

NH2N

NH

N

N

N O

NH2

N

B A

S E

S

DDNNAA

OH

P

O

HO

O

O

CH2

HO

O

H 2N

NHN N

N H

H

P HO

O

O

CH2

OO

N

O

H 2N

NH

H2O

H OH

P

O

HO

O

O

CH2

CH 3

O

O

HNN

H2O

5’Phosphate group

3’Hydroxyl group

5’Phosphategroup

3’Hydroxyl group


The Watson - Crick The Watson - Crick Model Of DNAModel Of DNA

3.4 nm1 nm

0.34 nm

Majorgroove

Minorgroove

A T

T AG C

C G

C GG C

T A

A T

G CT A

A TC G

--

-

-

---

--

--

--

-

--

--

-

---

--

--

--

-

-


Forms of the Double HelixForms of the Double Helix

0.26 nm

2.8 nmMinorgroove

Majorgroove

C GA T

T AG C

C G

G CT A

A T

G CT A

A TC G

A T

G C

1.2 nm

A DNA

1 nm

Majorgroove

Minorgroove

A T

T AG C

C G

C G

G CT A

A T

G CT A

A TC G

0.34 nm

3.9 nm

B DNA

+34.7o Rotation/Bp11 Bp/turn

-30.0o Rotation/Bp12 Bp/turn

+34.6o Rotation/Bp10.4 Bp/turn

C GG C

G CC G

C G

G CG C

G CC G

G CC G

0.57 nm

6.8 nm

0.9 nm

Z DNA


..

©2000 Timothy G. Standish..

A-DNA:1. Large hole in center

2. Sugar phosphate backbone is at the edge 3. Bases are displaced

towards edge B-DNA-1. Bases in center (no

hole) 2. Phosphates at periphery Z-DNA-1. Bases present

throughout the matrix of the helix

2. No exclusive domains for either bases or backbone 3. Left hand helix


Biological SignificanceBiological Significance

A-DNA-occurs only in dehydrated samples of DNA, such as those used in crystallographic experiments, and possibly is also assumed by DNA-RNA hybrid helices and by regions of double-stranded RNA.

Z-DNA has been found, it is commonly believed to provide torsional strain relief (supercoiling) while DNA transcription occurs. The potential to form a Z-DNA structure also correlates with regions of active transcription


C-DNA:– Exists only under high dehydration conditions– 9.3 bp/turn, 0.19 nm diameter and tilted bases

D-DNA:– Occurs in helices lacking guanine– 8 bp/turn

E-DNA:– Like D-DNA lack guanine– 7.5 bp/turn

P-DNA:– Artificially stretched DNA with phosphate groups found inside

the long thin molecule and bases closer to the outside surface of the helix

– 2.62 bp/turn

Even More Forms Of DNAEven More Forms Of DNA

B-DNA appears to be the B-DNA appears to be the most common form most common form in in vivovivo. However, under . However, under some circumstances, some circumstances, alternative forms of DNA alternative forms of DNA may play a biologically may play a biologically significant role.significant role.


Certain DNA sequences adopt Certain DNA sequences adopt unusual structuresunusual structures

Palindrome: The term is applied to regions of DNA with inverted repeats of base sequence having twofold symmetry over two strands of DNA. Such sequences are self-complementary within each strand and therefore have the potential to form hairpin or cruciform (cross-shaped) structures



Mirror repeats :When the inverted repeat occurs within each individual strand of the DNA, the sequence is called a mirror repeat.

Mirror repeats do not have complementary sequences within the same strand and cannot form hairpin or cruciform structures.


..


keto-enol keto-enol tautomerism keto-enol tautomerism refers

to a chemical equlibrium between a keto form (a ketone or an aldehyde) and an enol (An alcohol)

In DNA, the nucleotide bases are in keto form.

Rare enol tautomers of the bases G and T can lead to mutation because of their altered base-pairing properties.


Triplex DNATriplex DNA Nucleotides participating in a Watson-

Crick base pair can form a number of additional hydrogen bonds, particularly with functional groups arrayed in the major groove. For example, a cytidine residue (if protonated) can pair with the guanosine residue of a G-C nucleotide pair.


Triplex DNATriplex DNA

The N-7, O6, and N6 of purines, the atoms that participate in the hydrogen bonding of triplex DNA, are often referred to as Hoogsteen positions, and the non-Watson-Crick pairing is called Hoogsteen pairing.

The triplexes form most readily within long sequences containing only pyrimidines or only purines in a given strand

Four DNA strands can also pair to form a tetraplex


..


H-DNAH-DNA A particularly exotic DNA structure, known

as H-DNA, is found in polypyrimidine or polypurine tracts that also incorporate a mirror repeat. A simple example is a long stretch of alternating T and C residues


H-DNAH-DNA


Structure of RNAStructure of RNA The single strand of RNA tends to assume a

right-handed helical conformation dominated by base stacking Interactions ,which are strongest between two purines

The purine-purine interaction is so strong that a pyrimidine separating two purines is often displaced from the stacking pattern so that the purines can interact


Structure of RNAStructure of RNA RNA can base-pair with complementary regions

of either RNA or DNA. For DNA: G pairs with C and A pairs with U ,however base pairing between G and U is fairly common in RNA.

Where complementary sequences are present, the predominant double-stranded structure is an A-form right-handed double helix.

Hairpin loops form between nearby self-complementary sequences.


..

short base sequences (such as UUCG) are often found at the ends of RNA hairpins and are known to form particularly tight and stable loops.

Additional structural contributions are made by hydrogen bonds that are not part of standard Watson-Crick base pairs. For example, the 2-hydroxyl group of ribose can hydrogen-bond with other groups.

rRNA has a characteristic secondary structure due to many intramolecular H-bonds


Structure Of t-RNAStructure Of t-RNA


Denaturation and RenaturationDenaturation and Renaturation Heating double stranded DNA can overcome the

hydrogen bonds holding it together and cause the strands to separate resulting in denaturation of the DNA

When cooled relatively weak hydrogen bonds between bases can reform and the DNA renatures

TACTCGACATGCTAGCACATGAGCTGTACGATCGTG

Double stranded DNA

TACTCGACATGCTAGCACATGAGCTGTACGATCGTG

Double stranded DNA

Renaturation

TACTCGACATGCTAGCAC

ATGAGCTGTACGATCGTG

Denatured DNA

Denaturat

ion

Single stranded DNA


Denaturation and RenaturationDenaturation and Renaturation DNA with a high guanine and cytosine content has relatively more

hydrogen bonds between strands This is because for every GC base pair 3 hydrogen bonds are made

while for AT base pairs only 2 bonds are made Thus higher GC content is reflected in higher melting or

denaturation temperature

Intermediate melting temperature

Low melting temperature High melting temperature67 % GC content -

TGCTCGACGTGCTCGACGAGCTGCACGAGC

33 % GC content -

TACTAGACATTCTAGATGATCTGTAAGATC

TACTCGACAGGCTAGATGAGCTGTCCGATC

50 % GC content -


Determination of GC ContentDetermination of GC Content Comparison of melting temperatures can be used to

determine the GC content of an organisms genome To do this it is necessary to be able to detect whether DNA

is melted or not Absorbance at 260 nm of DNA in solution provides a means

of determining how much is single stranded Single stranded DNA absorbs 260 nm ultraviolet light more

strongly than double stranded DNA does although both absorb at this wavelength

Thus, increasing absorbance at 260 nm during heating indicates increasing concentration of single stranded DNA


Determination of GC ContentDetermination of GC Content

OD260

0

1.0

65 70 75 80 85 90 95

Temperature (oC)

Tm = 85 oCTm = 75 oC

Double stranded DNA

Single stranded DNA

Relatively low GC content

Relatively high GC content

Tm is the temperature at which half the DNA is melted


GC Content Of Some GenomesGC Content Of Some Genomes

Phage T7 48.0 %

Organism % GC

Homo sapiens 39.7 %

Sheep 42.4 %

Hen 42.0 %

Turtle 43.3 %

Salmon 41.2 %

Sea urchin 35.0 %

E. coli 51.7 %

Staphylococcus aureus 50.0 %

Phage 55.8 %


HybridizationHybridization The bases in DNA will only pair in very specific ways, G with C and

A with T In short DNA sequences, imprecise base pairing will not be tolerated Long sequences can tolerate some mispairing only if -G of the

majority of bases in a sequence exceeds the energy required to keep mispaired bases together

Because the source of any single strand of DNA is irrelevant, merely the sequence is important, DNA from different sources can form double helix as long as their sequences are compatible

Thus, this phenomenon of base pairing of single stranded DNA strands to form a double helix is called hybridization as it may be used to make hybrid DNA composed of strands which came from different sources


HybridizationHybridization

DNA from source “Y”

TACTCGACAGGCTAG

CTGATGGTCATGAGCTGTCCGATCGATCAT

DNA from source “X”

TACTCGACAGGCTAG

HybridizationHybridization


HybridizationHybridization Because DNA sequences will seek out and hybridize with other

sequences with which they base pair in a specific way much information can be gained about unknown DNA using single stranded DNA of known sequence

Short sequences of single stranded DNA can be used as “probes” to detect the presence of their complimentary sequence in any number of applications including:– Southern blots– Northern blots (in which RNA is probed)– In situ hybridization– Dot blots . . .

In addition, the renaturation or hybridization of DNA in solution can tell much about the nature of organism’s genomes


Reassociation KineticsReassociation Kinetics An organism’s DNA can be heated in solution until it

melts, then cooled to allow DNA strands to reassociate forming double stranded DNA

This is typically done after shearing the DNA to form many fragments a few hundred bases in length. The larger and more complex an organisms genome is, the longer it will take for complimentary strands to bum into one another and hybridize

Rate of reassociation is proportional to concentration of the two homologus dissociated strands.

Reassociation follows second order kinetics: dt/dc = -kc2 , now integrate this equation


Reassociation KineticsReassociation Kinetics The following equation describes the second order

rate kinetics of DNA reassociation:

11 + kCot

=CCo

Concentration of single stranded DNA after time t

Initial concentration of single stranded DNA

Second order rate constant (the important thing is that it is a constant)

Co (measured in moles/liter) x t (seconds). Generally graphed on a log10 scale.

Cot1/2 is the point at which half the initial concentration of single stranded DNA has annealed to form double-stranded DNA


CCoot t 0.5 0.5 valuevalue

Cot0.5 value is proportional to complexity of the genome.

A plot of C/Co against Cot is called Cot curve and it provides information about

complexity of a genome.


Genome complexityGenome complexity Complexity is the minimum length of DNA

that contains a single copy of all the single reiterated sequences that are represented within the genome.

Complexity of a genome is equal to its molecular mass only if a genome has unique nucleotide sequences (repetitive sequences absent).


exampleexample # For a hypothetical DNA-1 having three

nucleotide sequences, N1, N2, N3. Molecular mass=N1+N2+N3 Complixity=N1+N2+N3

# For a hypothetical DNA-2 having 103 copies of N1 ,105 copies of N2 & 1 copy of N3.

Molecular mass= 103 N1+ 105 N2 + N3 Complixity=N1+N2+N3


Reassociation KineticsReassociation Kinetics

Fraction remaining single-stranded (C/Co)

0

0.5

10-4 10-3 10-2 10-1 1 101 102 103 104

Cot (mole x sec./l)

1.0

Higher Cot1/2 values indicate greater genome complexityCot1/2


Reassociation KineticsReassociation Kinetics

0.5

Fraction remaining single-stranded (C/Co)

010-4 10-3 10-2 10-1 1 101 102 103 104

Cot (mole x sec./l)

1.0

Eukaryotic DNA

Prokaryotic DNA

Repetitive DNA Unique

sequence complex DNA


Repetitive DNARepetitive DNAOrganism % Repetitive DNA

Homo sapiens 21 %

Mouse 35 %

Calf 42 %

Drosophila 70 %

Wheat 42 %

Pea 52 %

Maize 60 %

Saccharomycetes cerevisiae 5 %

E. coli 0.3 %

©2000 timothy g. standish structure and analysis of dna

Documents