CHAPTER 4, PART 1
DNA and RNA- MOLECULES OF HEREDITY
CHAPTER 4, PART 1: LECTURE TOPICS
1. DNA/RNA structures, nomenclature, shorthand conventions
2. DNA and RNA as genetic material
3. General properties of DNA Double Helix
4. Basic mechanism of DNA replication
5. Some important physical and chemical properties of DNA
Reminders from Chapter 25 lecturesNucleic acids (DNA and RNA) are polymers consisting of pentose sugars,
phosphates and bases
DNA and RNA are “Polynucleotides”Levene (1920 book): Tetranucleotide hypothesis of DNA structure
G C
AT
Incorrect!!
A,G,C,T 5`
3`5`
3`
3`
5`
2`
2`
3` 5` Phosphodiester bond
2’
2’
5` 3` polarity (direction of chain; L to R)
A,G,C,U
DNA and RNA are POLYNUCLEOTIDES (chains of nucleotides)
RNA/DNA Shorthand ConventionsA G C T
P P3`5`
Base
Sugar
P
A C3`
5`(OH)
3` 5` Phosphodiester bond
(A dinucleotide)
Nucleic Acids Shorthand Notations
Example: pACG
5`-P
or:-OH-PP-PPP
(or P, etc. if indicated)
Assume both ends are OH if not indicated
Trinucleotide: [pACG]
P
A C G
5` PP OH 3`
b and c are the same as a
(b) pApCpG
(c) pACG
(a)
a=b=c and you can draw full structure from each shorthand
5`
H
3`
A
C
G
Structure of Trinucleotide: [pACG]
CHAPTER 4, PART 1: LECTURE TOPICS
1. DNA/RNA structures, nomenclature, shorthand conventions
2. DNA and RNA as genetic material
3. General properties of DNA Double Helix
4. Basic mechanism of DNA replication
5. Some important physical and chemical properties of DNA
DNA and RNA as genetic material: Proofs
• Transformation with pure DNA (Pneumococcus; 1928,1944)
• Bacteriophage T2 transmits DNA, not protein (1952)
• Tobacco mosaic virus transmits RNA, not protein (1953)
• Retroviruses (RNA to DNA to RNA)
• Prions (PROTEINS - not DNA or RNA) as transmissible disease agents
Nucleic acids discovered (1870’s) – Crimean War bandages
Hershey-Chase (1952): show that DNA (not protein) transmits genetic information
Even in early 1950’s, still asking: Is a gene protein or DNA??
Hershey-Chase Experiment: Biological system[To ask: Are genes protein or DNA??]
DNA
E. coli cell
T2 bacteriophage(from Paris sewers)
Scenario:attach to cellinject DNAreproduce (new DNA and proteins)kill cellsrelease progeny (new) virusinfect more cells
new
BACTERIOPHAGE VIRUS LIFE CYCLE
(32P) - DNA (35S) - protein
Infect
Cell DNA (32P)
Shake off virus (35S)
New virus has:
30% of 32P (DNA)
<1% of 35S (protein)
Hershey-Chase experiment (1952):DNA not protein transmits genetic information
Tobacco Mosaic Virus (TMV)
An RNA virus
RNA as Genetic Material (RNA RNA)
Ex: Tobacco Mosaic Virus
Type 1 RNA and protein
(1970) Retroviruses: RNA in virus transmits genetic information via a DNA intermediate in infected cells
RNA DNA RNA
(makes a cDNAstrand)
(cuts the RNA
strand)
(makes 2nd
cDNAstrand)
DNADNA
Example: HIV
RNA DNARNA
SLOW BRAIN INFECTIONSScrapie (Czeckoslovakia) Sheep, GoatsBovine spongiform encephalopathy(BSE) Mad Cow disease (England)
Cattle
Transmissible mink encephalopathy Mink
Creutzfeldt-Jakob disease *Kuru, Gerstmann-Strauss syndrome
Humans *
Chronic wasting disease with spongiform encephalopathy
Captive Rocky mountain elk and mule deer
Spongiform encephalopathy Nyala gemsbok Domestic cat
PRIONS – Proteins that are transmissible agents [PrPc]
- Promiscuous host range and cause CNS diseases
* Choreographer Balanchine died from this.
PRIONS [NOT DNA or RNA] – Mode of Action
Twisted abnormal prionsinduce shape change in normal ones and they accumulate around neurons.
Elk brain tissue: Light spots show spongy areas where tissue has been destroyed.
Incidence of Chronic Wasting Disease among elk and deer (2002)
Arizona(www.gf.state.az.us/)
SUMMARY
2. DNA and RNA are genetic material
Transformation with DNA
T2 DNA not Protein
TMV RNA
PRIONS (Proteins are transmissible agent)
Prions are not DNA or RNA – not Genes!
CHAPTER 4, PART 1: LECTURE TOPICS
1. DNA/RNA structures, nomenclature, shorthand conventions
2. DNA and RNA as genetic material
3. General properties of DNA Double Helix
4. Basic mechanism of DNA replication
5. Some important physical and chemical properties of DNA
Lysed E. coli cell reveals chromosomal DNA
E. Coli chromosome size: 4.6x106 base pairs
DNA
X-ray diffraction from a hydrated DNA-B fiber.
The central cross is diagnostic of a helical structure. The strong arcs on the meridian arise from the stack of base pairs
Double helix
3.4 Å
SugarBase
DNA molecules are usually double helices
DNA Base Composition:A = T
G = C
% GC
Low
High(genes)
A = T G = C
= 1 = 1
DNA: usually double-stranded with antiparallel strands
G C
AT
5`
5`3`
3`G = C
A = T5’
3’5’
3’
Watson-Crick model B-DNAA space-filling model of B-DNA from x-ray data [Fig. 4.11a]
BasesMinor groove
Major groove
Deoxyribose/ phosphate backbone
BasesTop view, looking down the helical axis [Figs 4.11b and 4.13]
Right handed helix
10 bp/turn;3.4 A/bp
Hydrogen bonds between paired bases
GC
AT
Compare with a chapter 25 slide showing groups involved in base pairing
A-T base pair
(A-U) in RNA
2 H-bonds
G-C base pair
3 H-bonds
T A
GC
Ring N atom
C=O
C=O
NH2
NH2
3`
3`
Bases are parallel
Sugar
Sugar
Phosphate
Sugars are perpendicular
to the bases3 Base pair stack in DNA
SUMMARY
3. General properties of Watson-Crick DNA Double helix
Antiparallel strands
Right-handed helix
10 bp / helix turn, 3.4A/bp
Bases on inside and parallel
Bases perpendicular to the deoxyribose-P chain
A=T and G=C base pairs
4. Basic mechanism of DNA replication
CHAPTER 4, PART 1: LECTURE TOPICS
Buoyant density of DNA
Distance from axix of rotation
1.68 1.7 1.72Density (g/cc)
Use gradient of CsCl of same density range as DNA
Buoyant density of DNA
% GC
1.68 1.7 1.72
N14 DNA
N15 DNA
Density (g/cc)
Use gradient of CsCl of same density range as DNA
E. Coli cells grown in media that contains 14N- or 15N-NH4+
14N 15N
UV photo of DNA
Photo density
Centrifugal force or DNA buoyant density (g/cc) or CsCl concentration
DNA replication is semiconservative (1958)[Meselson and Stahl]
CsCl
14N 15N
15N/14N
14N 15N/14N
(2) 14N
15N
15N/14N
Start
1 cell division
Mix
15N
Half 15N/14Nand
half 14N/14N
Transfer to 14Nmedium
2 celldivisions
E.Coli DNA polymerase I requires:
1. All four dNTPs (dATP, dGTP, dCTP and dTTP)
2. A primer chain with a free 3`-OH end
3. A template strand to which the primer is base-paired
• Double-stranded DNA that is fully intact and lacking a free 3`-OH end will not be replicated (Ex: Intact circular DNA)
4. Mg2+
Draw a template-primer complex to learn how it works.
DNA replication
DNA synthesis: DNA Polymerase Reaction
(DNA)n + dNTP (DNA)n+1 + PPi 2Pi
Primer 5` n+1 3` 5` n+2 3`
Template
2Pi 2Pi
DNA chain growth is 5’ to 3’
DNA Polymerase Reaction Mechanism:A nucleophilic attack of the 3`-OH on the α–P of dNTP
3`
5`
Nucleophilic attack New 3` 5` phosphodiester bond
3`
5`
αβγ
DNA REPLICATIONDNA template-primer complex for DNA polymerase
3`
5` 3`-OH
G A
dCTP dTTP…..etc
1 2
Accuracy: 1 “permanent” mistake every 108 bases
PPi
2Pi
C T
Template DNA
Primer DNA
5`
Direction of chain growth 5` 3`
Summary of basic mechanism of DNA replication
Replication is semiconservative
DNA polymerase requires a template-primer complex
dNTPs are the substrates for DNA synthesis
PPi breakdown to 2 Pi (catalyzed by pyrophosphatase) drives DNA synthesis
DNA Polymerase accuracy: 1 mistake every 108 bases
CHAPTER 4, PART 1: LECTURE TOPICS
5) Some important physical and
chemical properties of DNA
Some important physical-chemical properties of DNA
Buoyant density of DNA is proportional to %G-C bp
Reversible separation-reassociation of DNA strandsDenature/Renature/melting temperature (Tm) proportional to
%G-C bp)
Enormous range of lengths [:m to cm lengths]
Conformation of DNA can be:
Linear
circle (open/supercoiled)
single (SS) or double- stranded (DS)
Buoyant density of DNA
% GC
1.68 1.7 1.72
N14 DNA
Density (g/cc)
Use gradient of CsCl of same density range as DNA
Why would a G-C base pair have a higher density than an A-T base pair?
A-T base pair
(A-U) in RNA
2 H-bonds
G-C base pair
3 H-bonds
T A
GC
Ring N atom
C=O
C=O
NH2
NH2
Heat
Cool
DNA reversible strand separation
Melting (denature; break H-bonds)
Reannealing: renature; reform H-bonds (zipper analogy)
+ordered structure
disordered structure
(<A260) (>A260)
U.V. light
absorbance
“Ordered” (Native d.s.)
(Denatured) disordered”
50% of base pairs “broken”(denatured)
Melting of DNA is reversible
A-T rich
G-C rich
Tm (50% base paired)
A-T pairs melt first
DNA melting temperature
G-C pairs more stable
1 kb (kilobase) of DNA or RNA = 1000 bases single-stranded (1 kb) or double-stranded (1 kbp). [p=pair]
1 kb double-stranded DNA = 0.34 :m long
1 kb double-stranded DNA = 660 kd [660 d/base pair]
DNA Size Definitions:
ENORMOUS SIZE RANGES OF DNA GENOMES
1.7 micron (circle)
1.36 mm (circle)
99 cm (linear)
CIRCULAR DNAs
Open
Supercoiled
(E. coli)
Other circular DNA’s:
Plasmids
Bacterial Chromosomes
Chloroplasts
Mitochondria
SUMMARY
1. DNA/RNA structures
Know nomenclature + shorthand conventions
SUMMARY
2. DNA and RNA as genetic material
Transformation with DNA
T2 DNA not Protein
TMV RNA
PRIONS (Proteins as transmissible agent)
SUMMARY
3. General properties of Watson-Crick DNA Double helix
Antiparallel strands
Right-handed helix
10 bp / helix turn, 3.4A/bp
Bases on inside and parallel
Bases perpendicular to the deoxyribose-P chain
A=T and G=C base pairs
SUMMARY
4. Basic mechanism of DNA replication
Semiconservative
DNA polymerase I (template-primer complex)
PPi 2Pi drives DNA synthesis
SUMMARY
5. Some important physical-chemical properties of DNA
Buoyant density (proportional to %G-C)
Denature-renature-melting (Tm proportional to %G-C)
:m to cm lengths
Linear, circles (open/supercoiled), SS, DS
If lined up end to end, How long a line would All the DNA in your body make??
To:
Phoenix
Los Angeles
Moon and Back
Pluto and Back
Sun and Back 6 times
Around the world
One way to Mars
If lined up end to end, How long a line would All the DNA in your body make??
To:
Phoenix
Los Angeles
Moon and Back
Pluto and Back
Sun and Back 6 times
Around the world
One way to Mars