chapter 4, part 1 - university of...

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]

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!

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

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


% GC

1.68 1.7 1.72

N14 DNA

N15 DNA

Density (g/cc)


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


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)


% GC

1.68 1.7 1.72

N14 DNA

Density (g/cc)


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


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


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

chapter 4, part 1 - university of...

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