DNA

1
DNA RNA- Nucleic Acids and Protein Synthesis

• IB Biology
• Ch. 16 Campbell
• Ch. 6 Orange Book

2
Objectives

• Describe the history behind the discovery of DNA
  and its function
• Outline the structure of a nucleotide
• Describe the structure of the DNA molecule
• Describe the process of DNA replication including
  the various enzymes and that it is a
  semi-conservative process.

3
Introduction

• Your genetic endowment is the DNA you inherited
  from your parents.
• Nucleic acids are unique in their ability to
  direct their own replication.
• The resemblance of offspring to their parents
  depends on the precise replication of DNA and its
  transmission from one generation to the next.
• Once T.H. Morgans group showed that units of
  heredity are located on chromosomes, the two
  constituents of chromosomes - proteins and DNA -
  were the candidates for the genetic material.
• Until the 1940s, the great heterogeneity and
  specificity of function of proteins seemed to
  indicate that proteins were the genetic material.
• However, this was not consistent with experiments
  with microorganisms, like bacteria and viruses.

4
Discovery of DNA

• 1868 Miescher first isolated deoxyribonucleic
  acid, or DNA, from cell nuclei

5
Fredrick Griffith- 1928

• First suggestion that about what genes are made
  of.
• Worked with
• 1) Two strains of Pneumococcus bacteria
• Smooth strain (S) Virulent
  (harmful)
• Rough strain (R)
  Non-Virulent
• 2) Mice-were injected with these strains of
  bacteria and watched to see if the survived.
• 3) Four separate experiments were done
• -injected with rough strain (Lived)
• -injected with smooth strain (Died)
• -injected with smooth strain that was heat killed
  (Lived)
• -injected with rough strain heat killed smooth
  (????)

6
Mixture of heat-killed S cells and living R
cells
EXPERIMENT
Living R cells (control)
Living S cells (control)
Heat-killed S cells (control)
RESULTS
Mouse dies
Mouse healthy
Mouse dies
Mouse healthy
Living S cells
7
Griffiths Conclusion

• Somehow the heat killed smooth bacteria changed
  the rough cells to a virulent form.
• These genetically converted strains were called
  Transformations
• Something (a chemical) must have been transferred
  from the dead bacteria to the living cells which
  caused the transformation
• Griffith called this chemical a Transformation
  Principle

8
Next Breakthrough came from the use of Viruses
T2 Bacteriophage- a typical virus

• Viruses provided some of the earliest evidence
  that genes are made of DNA
• Molecular biology studies how DNA serves as the
  molecular basis of heredity
• Are only composed of DNA and a protein shell.

9
Phage reproductive cycle
Phage attaches to bacterial cell.
Phage injects DNA.
Phage DNA directs host cell to make more phage
DNA and protein parts. New phages assemble.
Cell lyses and releases new phages.
Figure 10.1C
10
Photo of T2 Viruses
Fig. 16-3
Phage head
Tail sheath
Tail fiber
DNA
100 nm
Bacterial cell
11
Hershey-Chase Experiment- 1952

• In 1952, Alfred Hershey and Martha Chase
  performed experiments showing that DNA is the
  genetic material of a phage known as T2
• To determine the source of genetic material in
  the phage, they designed an experiment showing
  that only one of the two components of T2 (DNA or
  protein) enters an E. coli cell during infection
• They concluded that the injected DNA of the phage
  provides the genetic information

12
Fig. 16-4-1
EXPERIMENT
Radioactive protein
Phage
Bacterial cell
DNA
Batch 1 radioactive sulfur (35S)
Radioactive DNA
Batch 2 radioactive phosphorus (32P)
13
Fig. 16-4-2
EXPERIMENT
Empty protein shell
Radioactive protein
Phage
Bacterial cell
DNA
Batch 1 radioactive sulfur (35S)
Phage DNA
Radioactive DNA
Batch 2 radioactive phosphorus (32P)
14
Fig. 16-4-3
EXPERIMENT
Empty protein shell
Radioactivity (phage protein)
in liquid
Radioactive protein
Phage
Bacterial cell
DNA
Batch 1 radioactive sulfur (35S)
Phage DNA
Centrifuge
Pellet (bacterial cells and contents)
Radioactive DNA
Batch 2 radioactive phosphorus (32P)
Centrifuge
Radioactivity (phage DNA) in pellet
Pellet
15
Video Clip of Hershey-Chase

• http//highered.mcgraw-hill.com/sites/0072437316/s
  tudent_view0/chapter14/animations.html

16
Erwin Chargaff- 1950

• Already known- DNA is a polymer of nucleotides-
  nitrogen base, pentose sugar, and a phosphate
  group.
• Chargaff noticed a ratio of the bases
• 30.3 Adenine
• 30.3 Thymine
• 19.5 Guanine
• 19.9 Cytosine
• So, for DNA, the amt. of A T, and
• The amt. of C G (Chargaffs Rules)

17
Who Discovered the Shape of DNA?

• James Watson and Francis Crick (1954) are
  credited with finally piecing together all the
  information previously gathered on the molecule
  of DNA. They established the structure as a
  double helix - like a ladder that is twisted. The
  two sides of the ladder are held together by
  hydrogen bonds.

18
How did they get to their conclusions?

• They built many models, always perplexed at how
  it fit together, until one day, when they
  wandered into the office of a fellow scientist,
  Dr. Rosalind Franklin.

19
Along with Dr. Maurice Wilkins, she had taken
x-ray crystallography photos of DNA. They saw
her photos and realized the great secret- that
DNA was coiled like a spring. They then made
their model and won the Nobel Prize in 1962.
Franklins famous photo of DNA
20
So, What is DNA?Deoxyribonucleic Acid

• blueprint of life (has the instructions for
  making an organism)
• codes for your genes
• made of repeating subunits called nucleotides
• shape is the double helix (twisted ladder)

21
I. Structure of DNA- 3 parts

• Sugar- Deoxyribose
• Phosphate Group
• Nitrogen bases
• The sugar and phosphates make up the "backbone"
  of the DNA molecule.

Nucleotide
22
DNA and RNA are polymers of Nucleotides
DNA is a nucleic acid, made of long chains of
nucleotides- Sugar, phosphate, nitrogen base.
Phosphate group
Nitrogenous base
Nitrogenous base(A, G, C, or T)
Sugar
Phosphategroup
Nucleotide
Thymine (T)
Sugar(deoxyribose)
DNA nucleotide
Figure 10.2A
Polynucleotide
Sugar-phosphate backbone
23
Nitrogen bases

• Bases come in two types
• a. Purines (adenine and guanine- AG)
• b. Pyrimidines (thymine and cytosine- TC).

24
DNA Maintains a Uniform Diameter

• See pg. 310

25
Base Pairing- Chargaffs Rules

• Watson and Crick reasoned that the pairing was
  more specific, dictated by the base structures
• They determined that adenine (A) paired only with
  thymine (T), and guanine (G) paired only with
  cytosine (C)
• The Watson-Crick model explains Chargaffs rules
  Adenine pairs to Thymine (A-T)Guanine pairs
  to Cytosine (G-C)Very important- remember
  this!!!!

26
DNA Bonding
The two sides of the helix are held together by
Hydrogen bonds (weak) The sides of the DNA, the
sugar and phosphate, are held together with
covalent bonds.
27
Simple Diagram of DNA-Think of it like a ladder,
the bases being the rungs.
28

• Each strand of the double helix is oriented in
  the opposite direction
• The ends are referred to as the 3 and 5 ends.

5? end
3? end
P
P
P
P
P
P
P
P
3? end
5? end
Figure 10.5B
29

• Summary
• Chargaff ratio of nucleotide bases (AT CG)
• Watson Crick (Wilkins, Franklin)
• The Double Helix
• v nucleotides nitrogenous base (thymine,
  adenine, cytosine, guanine) sugar deoxyribose
  phosphate group

30
Putting it all together here are some images of
what the DNA double helix looks like
31
DNA Replication and Repair

• The relationship between structure and function
  is manifest in the double helix
• Watson and Crick noted that the specific base
  pairing suggested a possible copying mechanism
  for genetic material

32
The Basic Principle Base Pairing to a Template
Strand

• Since the two strands of DNA are complementary,
  each strand acts as a template for building a new
  strand in replication
• In DNA replication, the parent molecule unwinds,
  and two new daughter strands are built based on
  base-pairing rules

33
Fig. 16-9-1
A
T
C
G
T
A
T
A
C
G
(a) Parent molecule
34
Fig. 16-9-2
A
T
T
A
C
G
G
C
A
T
A
T
T
T
A
A
C
C
G
G
(b) Separation of strands
(a) Parent molecule
35
Fig. 16-9-3
A
A
T
T
A
T
T
A
C
C
G
G
G
C
G
C
A
T
A
A
T
A
T
T
T
T
A
T
T
A
A
A
C
C
G
C
C
G
G
G
(c) Daughter DNA molecules, each consisting of
one parental strand and one new strand
(b) Separation of strands
(a) Parent molecule
36

• Watson and Cricks semiconservative model of
  replication predicts that when a double helix
  replicates, each daughter molecule will have one
  old strand (derived or conserved from the
  parent molecule) and one newly made strand
• Competing models were the conservative model (the
  two parent strands rejoin) and the dispersive
  model (each strand is a mix of old and new)

37
Three Proposed Models of DNA Replication

• 
  
  

38
DNA Replication A Closer Look

• The copying of DNA is remarkable in its speed and
  accuracy
• More than a dozen enzymes and other proteins
  participate in DNA replication

39
DNA replication depends on specific base pairing

• In DNA replication, the strands separate
• Enzymes use each strand as a template to assemble
  the new strands

Nucleosomes
Parental moleculeof DNA
Both parental strands serveas templates
Two identical daughtermolecules of DNA
40
Anti-parallel Structure of DNA
41
Antiparallel nature

• 5 end corresponds to the Phosphate end
• 3 end corresponds to the OH sugar
• Replication runs in BOTH directions
• One strand runs 5 to 3 while the other runs
  3 to 5
• Nucleotides are added on the 3 end of the
  original strand.
• The new DNA strand forms and grows in the 5 ?
  3 direction only

42
Building New Strands of DNA
5 end
3 end
5 end
43
Building New Strands of DNA

• Each nucleotide is a triphosphate
• (GTP, TTP, CTP, and ATP)
• Nucleotides only add to the 3 end of the growing
  strand (never on the 5 end)
• Two phosphates are released (exergonic) and the
  energy released drives the polymerization process.

44
Getting Started

• Replication begins at special sites called
  origins of replication, where the two DNA strands
  are separated, opening up a replication bubble
• A eukaryotic chromosome may have hundreds or even
  thousands of origins of replication
• Replication proceeds in both directions from each
  origin, until the entire molecule is copied

45
Fig. 16-12b
Origin of replication
Double-stranded DNA molecule
Parental (template) strand
Daughter (new) strand
0.25 µm
Replication fork
Bubble
Two daughter DNA molecules
(b) Origins of replication in eukaryotes
46
Fig. 16-12
Origin of replication
Parental (template) strand
Daughter (new) strand
Replication fork
Double- stranded DNA molecule
Replication bubble
0.5 µm
Two daughter DNA molecules
(a) Origins of replication in E. coli
Origin of replication
Double-stranded DNA molecule
Parental (template) strand
Daughter (new) strand
0.25 µm
Replication fork
Bubble
Two daughter DNA molecules
(b) Origins of replication in eukaryotes
47
Getting Started- Enzymes

• At the end of each replication bubble is a
  replication fork, a Y-shaped region where new DNA
  strands are elongating
• Helicases are enzymes that untwist the double
  helix at the replication forks
• Single-strand binding protein binds to and
  stabilizes single-stranded DNA until it can be
  used as a template
• Topoisomerase corrects overwinding ahead of
  replication forks by breaking, swiveling, and
  rejoining DNA strands

48
Fig. 16-13
Primase
Single-strand binding proteins
3?
Topoisomerase
5?
3?
RNA primer
5?
5?
3?
Helicase
49

• DNA polymerases cannot initiate synthesis of a
  polynucleotide they can only add nucleotides to
  the 3? end
• The initial nucleotide strand is a short RNA
  primer

50
RNA Primers

• An enzyme called primase can start an RNA chain
  from scratch and adds RNA nucleotides one at a
  time using the parental DNA as a template
• The primer is short (510 nucleotides long), and
  the 3? end serves as the starting point for the
  new DNA strand.

51
Synthesizing a New DNA Strand

• Enzymes called DNA polymerases catalyze the
  elongation of new DNA at a replication fork
• Most DNA polymerases require a primer and a DNA
  template strand
• The rate of elongation is about 500 nucleotides
  per second in bacteria and 50 per second in human
  cells

52
3?
DNA polymerasemolecule
5?
How DNA daughter strands are synthesized
5? end
Daughter strandsynthesizedcontinuously
Parental DNA
5?
3?
Daughter strandsynthesizedin pieces
3?
P
5?

• The daughter strands are identical to the parent
  molecule

5?
P
3?
DNA ligase
Overall direction of replication
Figure 10.5C
53
Laying Down RNA Primers
54
DNA Replication-New strand Development

• Leading strand synthesis is toward the
  replication fork
• (only in a 5 to 3 direction from the 3
  to 5 master strand)
• -Continuous
• Lagging strand synthesis is away from the
  replication fork
• -Only short pieces are made called Okazaki
  fragments
• - Okazaki fragments are 100 to 2000 nucleotides
  long
• -Each piece requires a separate RNA primer
• -DNA ligase joins the small segments together
• (must wait for 3 end to open again in a
  5 to 3 direction)
• View video clip
• http//highered.mcgraw-hill.com/sites/0072437316/s
  tudent_view0/chapter14/animations.html

55
DNA Replication Fork
56
Fig. 16-16a
Overview
Origin of replication
Leading strand
Lagging strand
Lagging strand
2
1
Leading strand
Overall directions of replication
57
Video Clip of DNA Replication

• http//highered.mcgraw-hill.com/sites/0072437316/s
  tudent_view0/chapter14/animations.html

58
Key Enzymes Required for DNA Replication (pg. 314)

• Helicase - catalyzes the untwisting of the DNA at
  the replication fork
• SSBPs - single stranded binding proteins,
  prevents the double helix from reforming
• Topoisomerase Breaks the DNA strands and
  prevents excessive coiling
• Primase synthesizes the RNA primers and starts
  the replication first by laying down a few
  nucleotides initially.
• DNA Polymerase III - catalyzes the elongation
  of new DNA and adds new nucleotides on the 3 end
  of the growing strand.
• DNA polymerase I- Replaces the RNA primers.
• Ligase- Connects the Okazaki fragments.

59
Prokaryotic vs Eukaryotic Replication

• Prokaryotes
• Circular DNA (no free ends)
• Contains 4 x 106 base pairs (1.35 mm)
• Only one origination point
• Eukaryotes
• -Have free ends
• -Contains 3 x 109 base pairs (haploid cells) 1
  meter
• -Lagging strand is not completely replicated
• -Small pieces of DNA are lost with every cell
  cycle
• -End caps (Telomeres) protect and help to retain
  the genetic information

60
Issues with Replication

• Prokaryotes (ex. E. coli)
• Have one singular loop of DNA
• E. coli has approx. 4.6 million Nucleotide base
  pairs
• Rate for replication 500 nucleotides per second
• Eukaryotes w/Chromosomes
• Each chromosome is one DNA molecule
• Humans (46) has approx. billion base pairs
• Rate for replication 50 per second (humans)
• Errors
• Rate is one every 10 billion nucleotides copied
• Proofreading is achieved by DNA polymerase (pg.
  318)

61
Proofreading and Repairing DNA

• DNA polymerases proofread newly made DNA,
  replacing any incorrect nucleotides
• In mismatch repair of DNA, repair enzymes correct
  errors in base pairing
• DNA can be damaged by chemicals, radioactive
  emissions, X-rays, UV light, and certain
  molecules (in cigarette smoke for example)
• In nucleotide excision repair, a nuclease cuts
  out and replaces damaged stretches of DNA

62
Proofreading and Repairing DNA

1. Thymine dimer distorts the DNA molecule
2. A nuclease enzyme cuts the damaged DNA strand at
   two points and the damaged section is removed.
3. Repair synthesis by a DNA polymerase fills in the
   missing nucleotides.
4. DNA ligase seals the free end of the new DNA to
   the old DNA, making the strand complete.

Nuclease
Nuclease
DNA polymerase
DNA polymerase
DNA ligase
DNA ligase
63
Telomeres

• Short, non-coding pieces of DNA
• Contains repeated sequences (ie. TTGGGG 20
  times)
• Can lengthen with an enzyme called Telomerase
• Lengthening telomeres will allow more
  replications to occur.
• Telomerase is found in cells that have an
  unlimited number of cell cycles (commonly
  observed in cancer cells)
• Artificially giving cells telemerase can induce
  cells to become cancerous
• Shortening of these telomeres may contribute to
  cell aging and Apotosis (programmed cell death)
• Ex. A 70 yr old persons cells divide approx.
  20-30X vs an infant which will divide 80-90X

64
Fig. 16-20
Telomeres
1 µm
65
A chromosome consists of a DNA molecule packed
together with proteins

• The bacterial chromosome is a double-stranded,
  circular DNA molecule associated with a small
  amount of protein
• Eukaryotic chromosomes have linear DNA molecules
  associated with a large amount of protein
• In a bacterium, the DNA is supercoiled and
  found in a region of the cell called the nucleoid

66
Chromatin Packing In Eukaryotes

• Chromatin is a complex of DNA and protein, and is
  found in the nucleus of eukaryotic cells
• Histones are proteins that are responsible for
  the first level of DNA packing in chromatin
• Nucleosomes- are like beads on a string, consist
  of DNA wound twice around a protein core composed
  of two molecules each of the 4 main histone
  types.

67
Fig. 16-21a
Nucleosome (10 nm in diameter)
DNA double helix
(2 nm in diameter)
H1
Histone tail
Histones
DNA, the double helix
Histones
Nucleosomes, or beads on a string (10-nm fiber)
See p. 320
68
Fig. 16-21b
Chromatid (700 nm)
30-nm fiber
Loops
Scaffold
300-nm fiber
Replicated chromosome (1,400 nm)
30-nm fiber
Looped domains (300-nm fiber)
Metaphase chromosome
69
(No Transcript)
70
Part 2

• RNA

71
I. Structure of RNA- 3 parts

• Sugar- Ribose
• Phosphate Group
• Nitrogen-containing bases
• Adenine
• Thymine
• Guanine
• Uracil (substituted for Cytosine)
• Note- RNA is single-stranded, unlike DNA.

72
3 Types of RNA (used to build proteins)

1. Messenger RNA (mRNA)- carries the instructions
   from DNA to the ribosomes.
2. Transfer RNA (tRNA)- carries message from mRNA to
   find the specific amino acids.
3. Ribosomal RNA (rRNA)- makes up ribosomes, it puts
   the proteins together.

73
Look at these 3 like parts of a construction site
Messenger RNA (mRNA) is the blueprint for
construction of a protein. Ribosomal RNA (rRNA)
is the construction site where the protein is
made. Transfer RNA (tRNA) is the truck
delivering the proper amino acid to the site at
the right time.
74
B. Transcription- The copying of information from
DNA to RNA

• RNA is very similar to DNA with the following
  exceptions
• it is single stranded
• it has uracil instead of thymine
• it has the sugar ribose, instead of deoxyribose
• The base-pair rule is followed during
  transcription, except, instead of pairing thymine
  with adenine, when creating an RNA strand, uracil
  is used
• DNA Strand T G C A T C A G A
• RNA Strand A C G U A G U C U

75
Transcription begins on the area of DNA that
contains the gene. Each gene has three
regions 1. Promotor - turns the gene on or
off2. Coding region - has the information on how
to construct the protein3. Termination sequence
- signals the end of the gene RNA Polymerase is
responsible for reading the gene, and building
the mRNA strand.
76

• How it works
• DNA unzips, exposing codons (3 bases in a row).
• RNA nucleotides move in (U to A, A to T, C to G,
  G to C) to form mRNA codons.
• mRNA strand pulls away, leaves nucleus.
• nRNA goes to ribosome (made of rRNA)
• Look at this as the writing of the code, or
  plans. The plans are at the construction site
  and now the bricks (amino acids) must be
  assembled according to the plan from the DNA.

77
Protein Synthesis- Translation

• Proteins are polymers, made of polypeptides.
• Each is made with a specific sequence of amino
  acids

78
The Genetic Code

• The genetic code is used to translate mRNA into
  proteins.
• Amino acids exist freely in the cytoplasm, many
  of which you acquire from your diet.

79
Codons

• Each 3 bases of RNA is called a codon, which
  translate to a single amino acid. (See codon
  chart).
• AUG is the start codon. This tells the ribosome
  to start making proteins.

80
Codon Chart- AUG is the start codon
Test for Understanding A DNA sequence has the
following bases T A C - A G A - T T A - G G G -
A T T What amino acids does it code for? (You'll
need to use the codon chart)
81
Translation

1. The mRNA travels to the cytoplasm.
2. The ribosome looks for the "start" codon - AUG,
   this is where the chain begins.
3. Transfer RNA (tRNA), has an anticodon at one end
   and an amino acid at the other, it binds to a
   complementary codon.

82

• 4. Another tRNA reads the next codon, the amino
  acid attached to it binds with the amino acid on
  the previous tRNA using a peptide bond. The first
  tRNA falls off.
• 5. This process continues until the "stop" codon
  is reached.

83
6. The amino acid chain folds into a 3
dimensional structure, now a protein. 7. A
releasing factor causes the ribosome to release
the protein.
84
Translation Diagram
85
Summary

• DNA Transcription RNA Translation Protein
• DNA RNA
• Only 1 type 3 types
• Deoxyribose ribose
• A,C,G,T A,C,G,U
• In nucleus in nucleus cytoplasm
• Made by replication made by transcription-mRNA
• DNA codons RNA codons anticodons

86
Online animations

• Protein Synthesis
• http//www.wisc-online.com/objects/index_tj.asp?ob
  jidAP1302
• Transcription to Translation
• http//207.207.4.198/pub/flash/26/transmenu_s.swf