Genetics: Analysis and Genetics: Analysis and PrinciplesPrinciples

CHAPTER 9CHAPTER 9

Molecular structure of DNA and Molecular structure of DNA and RNARNA

By Robert J. BrookerBy Robert J. Brooker

Roles of the genetic material

“The genetic material must carry out two jobs: duplicate

itself and control the development of the rest of the cell in a specific way”

Francis Crick

To fulfill its role, the genetic material must meet several criteria

1. Information: It must contain the information necessary to make an entire organism

2. Transmission: It must be passed from parent to offspring

3. Replication: It must be copied

In order to be passed from parent to offspring

4. Variation: It must be capable of changes

To account for the known phenotypic variation in each species

Miescher Discovered DNAMiescher Discovered DNA

18681868 Johann Miescher investigated the chemical Johann Miescher investigated the chemical

composition of the nucleuscomposition of the nucleus Isolated an organic acid that was high in Isolated an organic acid that was high in

phosphorusphosphorus He called it nucleinHe called it nuclein We call it DNA (deoxyribonucleic acid)We call it DNA (deoxyribonucleic acid)

Mystery of the Mystery of the Hereditary MaterialHereditary Material

Originally,they thought this was a proteinOriginally,they thought this was a protein The underlying idea was: The underlying idea was:

hereditary characteristics are very varioushereditary characteristics are very various The resposible molecules should be various The resposible molecules should be various

tootoo Peptides conist of 20 amino acids and Peptides conist of 20 amino acids and

therefore they are various in structure. therefore they are various in structure.

Discovery of a “transforming Discovery of a “transforming principle”principle”

Frederick Griffith, 1928Frederick Griffith, 1928

Pneumonia (Pneumonia (Diplococcus pneumoniaeDiplococcus pneumoniae) infecteren muizen. ) infecteren muizen.

Muizen krijgen longontsteking en sterven .Muizen krijgen longontsteking en sterven .

Twee type bacteriën :Twee type bacteriën :

R bacterie “rough coat”: - geen ontstekingR bacterie “rough coat”: - geen ontsteking

S bacterie “smooth coat”- wel ontstekingS bacterie “smooth coat”- wel ontsteking

““Coat type is associated with virulence”.Coat type is associated with virulence”.

Healthy Mouse

Injection

Bacterialcolonies

Rough nonvirulent(strain R)

Results

Mouse healthy

Smooth virulent(strain S)

Mouse dies

Heat-killedsmooth virulent(strain S)

Live strain S bacteriain blood samplefrom dead mouse

Mouse diesMouse healthy

+

Rough nonvirulent(strain R)

Heat-killedsmooth virulent(strain S)

Griffith’s experiment identifying the “transforming principle”

What is the “transforming What is the “transforming principle”?principle”?

Conclusion: DNA is the transforming principle allowing R Conclusion: DNA is the transforming principle allowing R

bacteria to make a smooth coat and allow infection.bacteria to make a smooth coat and allow infection.

Oswald Avery, Colin MacLeod and Maclyn McCarty, 1944

Heat-killed S bacteria “transformed” the R bacteria to a form that causes pneumonia

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Figure 9.3 Avery et al also conducted the following experiments

To further verify that DNA, and not a contaminant (RNA or protein), is the genetic material

05_04_Avery_MacLeod.jpg05_04_Avery_MacLeod.jpg

Hershey and Chase ResultsHershey and Chase Results

BacteriophagesBacteriophages

Viruses that infect Viruses that infect bacteriabacteria

Consist of protein Consist of protein and DNAand DNA

Inject their Inject their hereditary material hereditary material into bacteriainto bacteria

cytoplasm

bacterial cell wall plasma

membrane

virus particlelabeled with 35S

DNA (blue)being injected into bacterium

35S remainsoutside cells

virus particlelabeled with 32P

DNA (blue)being injected into bacterium

35P remainsinside cells

Fig 9b 225

Hershey and Chase ResultsHershey and Chase Results

In 1956, A. Gierer and G. Schramm isolated RNA In 1956, A. Gierer and G. Schramm isolated RNA from the tobacco mosaic virus (TMV), a plant virusfrom the tobacco mosaic virus (TMV), a plant virus Purified RNA caused the same lesions as intact TMV Purified RNA caused the same lesions as intact TMV

viruses viruses • Therefore, the viral genome is composed of RNATherefore, the viral genome is composed of RNA

Since that time, many RNA viruses have been Since that time, many RNA viruses have been foundfound

RNA Functions as the Genetic RNA Functions as the Genetic Material in Some VirusesMaterial in Some Viruses

Structure of the Structure of the Hereditary MaterialHereditary Material

Experiments in the 1950s showed that Experiments in the 1950s showed that DNA is the hereditary materialDNA is the hereditary material

But how does it look like?But how does it look like?

Scientists competed strongly to determine Scientists competed strongly to determine the structure of DNAthe structure of DNA

Structure of Nucleotides Structure of Nucleotides in DNAin DNA

Each nucleotide consists ofEach nucleotide consists of Deoxyribose (5-carbon sugar) Deoxyribose (5-carbon sugar)

Phosphate groupPhosphate group

A nitrogen-containing baseA nitrogen-containing base

Four basesFour bases Adenine, Guanine, Thymine, CytosineAdenine, Guanine, Thymine, Cytosine

DNA (DNA (ddeoxyriboeoxyribonnucleic ucleic aacid) and RNA cid) and RNA

((rriboibonnucleic ucleic aacid) are chains of nucleotidescid) are chains of nucleotides

Base - one of four types: adenine (A), thymine (T) or Base - one of four types: adenine (A), thymine (T) or uracil (U) in RNAuracil (U) in RNA

guanine (G), cytosine (C) guanine (G), cytosine (C)

Sugar - deoxyribose (DNA) or ribose (RNA)

Phosphate

Nucleotides are composed of:

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Base + sugar nucleoside Example

Adenine + ribose = AdenosineAdenine + deoxyribose = Deoxyadenosine

Base + sugar + phosphate(s) nucleotide Example

Adenosine monophosphate (AMP)Adenosine diphosphate (ADP)Adenosine triphosphate (ATP)

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Figure 9.9 The structure of nucleotides found in (a) DNA and (b) RNA

A, G, C or T

These atoms are found within individual nucleotides However, they are removed when nucleotides join together to make

strands of DNA or RNA

A, G, C or U

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or displayFigure 9.10

Base always attached here

Phosphates are attached there

Biochemical experiments of ChargaffBiochemical experiments of Chargaff

% adenine = % thymine % adenine = % thymine % guanine = % cytosine% guanine = % cytosine

% adenine = % thymine % adenine = % thymine % guanine = % cytosine% guanine = % cytosine

Complementary bases pair:Complementary bases pair:

A and T pairA and T pairC and G pairC and G pair

An explanation of the data : Model

She worked in the She worked in the same laboratory as same laboratory as Maurice WilkinsMaurice Wilkins

She used X-ray She used X-ray diffraction to study diffraction to study wet fibers of DNAwet fibers of DNA

Rosalind FranklinRosalind Franklin

The diffraction pattern is interpreted (using mathematical theory)

This can ultimately provide information concerning the

structure of the molecule

09_06.jpg09_06.jpg

James Watson

FrancisCrick

DNA nucleotide basesDNA nucleotide bases

phosphate group

deoxyribose

ADENINE (A)

THYMINE (T)

CYTOSINE (C)

GUANINE (G)

Figure 9.11

Phosphodiester bond

Watson-Crick ModelWatson-Crick Model DNA consists of two nucleotide strandsDNA consists of two nucleotide strands

The nucleotides are connected by phoshodiester The nucleotides are connected by phoshodiester

bondsbonds

The 2 strands run in opposite directionsThe 2 strands run in opposite directions

Strands are held together by hydrogen bonds between Strands are held together by hydrogen bonds between

basesbases

A binds with T and C with GA binds with T and C with G

Molecule is a double helixMolecule is a double helix

Esscher

AThe strands

areAnti-parallel

BThe strands

arecomplementary

CThe mechanism for copying lies within its structure

Three important features of DNA

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Nucleotides are covalently linked together by phosphodiester bonds A phosphate connects the 5’ carbon of one nucleotide to

the 3’ carbon of another

Therefore the strand has directionality5’ to 3’

The phosphates and sugar molecules form the backbone of the nucleic acid strand The bases project from the backbone

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Figure 9.17

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Figure 9.18

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To fit within a living cell, the DNA double helix must be extensively compacted into a 3-D conformation This is aided by DNA-binding proteins

The Three-Dimensional Structure The Three-Dimensional Structure of DNAof DNA

9-56Figure 9.21

DNA wound around histone

proteins

The DNA double helix can form different The DNA double helix can form different types of secondary structuretypes of secondary structure

The predominant form found in living cells is The predominant form found in living cells is B-DNA B-DNA

However, under certain However, under certain in vitroin vitro conditions, conditions, A-DNA A-DNA and and Z-DNAZ-DNA double helices can form double helices can form

DNA Can Form Alternative DNA Can Form Alternative Types of Double HelicesTypes of Double Helices

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A-DNA Right-handed helix 11 bp per turn Occurs under conditions of low humidity Little evidence to suggest that it is biologically important

Z-DNA Left-handed helix 12 bp per turn Its formation is favored by

GG-rich sequences, at high salt concentrations Cytosine methylation, at low salt concentrations

Evidence from yeast suggests that it may play a role in transcription and recombination

Bases substantially tilted relative to the central

axis

Bases substantially tilted relative to the central

axis

Sugar-phosphate backbone follows a

zigzag pattern

Bases relatively perpendicular to the

central axis

No details

Triplex DNA formation Triplex DNA formation is sequence specificis sequence specific

The pairing rules areThe pairing rules are

Triplex DNA has been Triplex DNA has been implicated in several implicated in several cellular processescellular processes Replication, Replication,

transcription, transcription, recombinationrecombination

Cellular proteins that Cellular proteins that specifically recognize specifically recognize triplex DNA have been triplex DNA have been recently discoveredrecently discovered

T binds to an AT pair in

biological DNA

C binds to a CG pair in

biological DNA

DNA can even form triple helixesDNA can even form triple helixes

The primary structure of an RNA strand is much The primary structure of an RNA strand is much like that of a DNA strandlike that of a DNA strand

RNA strands are typically several hundred to RNA strands are typically several hundred to several thousand nucleotides in lengthseveral thousand nucleotides in length

In RNA synthesis, only one of the two strands of In RNA synthesis, only one of the two strands of DNA is used as a templateDNA is used as a template

RNA StructureRNA Structure

Figure 9.22

RNAs can fold in to 3D structure:like proteins

“RNAs kink, bend, loop and twist themselves into a wonderfulvariety of shapes” (Doudna, Nature 388, 1997)

- RNAs lack the chemical diversity of proteins

but

- Many conformational degrees of freedom of its

phosphate backbone

- Unique hydrogen-bonding

- stacking of nucleotide bases

>> Ample resources for 3D-folding:

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Although usually single-stranded, RNA molecules can form short double-stranded regions This secondary structure is due to complementary base-

pairing A to U and C to G

This allows short regions to form a double helix

RNA double helices typically Are right-handed Have the A form with 11 to 12 base pairs per turn

Different types of RNA secondary structures are possible

Many factors Many factors contribute to the contribute to the tertiary structure of tertiary structure of RNARNA For exampleFor example

• Base-pairing and Base-pairing and base stacking within base stacking within the RNA itselfthe RNA itself

• Interactions with Interactions with ions, small ions, small molecules and large molecules and large proteinsproteins

Molecule contains single- and double-stranded regions

These spontaneously interact to produce this 3-D structure

Figure 9.23

Also called hair-pin

Complementary regions

Noncomplementary regions

Held together by hydrogen bonds

Have bases projecting away from double stranded regions

Example: 3D structure of tRNA-Phe from yeast

Hairpin Loops

Stems

Bulge loop

Interior loops

Multi-branched loop

RNAs are folded into complex structures becauseof their ability to form internal duplexes

M.McManus

Divalent cations, Mg++, are essential for correct folding, stability, and catalysis. (example: >100 Mg2+ ions needed for the folding of RNAse P

1. Identification of DNA as the Genetic Material

1. Experiments with pneumococcus suggested that DNA is the genetic material

2. Hershey and Chase provided evidence that the genetic material injected into the bacterial cytoplasm is T2 phage DNA

3. RNA functions as the genetic material in some viruses

2. Nucleic acid structure

1. Nucleotides are the building blocks of nucleic acids

2. Nucleotides are linked together to form a strand

3. A few key events led to the discovery of the double helix structure

4. Chargaff found that DNA has a biochemical composition in which the amount of A equals T and the amount of G equals C

5. Watson and Crick deduced the double helical structure of DNA

6. The molecular structure of the DNA double helix has several key features

7. DNA can form alternative types of double helices

8. DNA can form a triple helix, called triplex DNA

9. The three-dimensional structure of DNA within chromosomes requires additional folding and the association with proteins

10. RNA molecules are composed of strands that fold into specific structures

Summary : Outline of this chapter 9