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Tutorial - DNA
Watson & Crick
Griffith’s Experiments (Streptococcus pneumoniae)
Experiment
Results
Conclusion
1
R-Strain bacteria
S-Strain bacteria
Mouse lived
Mouse died
Nonvirulent strain of bacteria
Virulent/deadly strain
2
Heat-killed S-Strain
Mouse lived
Destroyed deadly bacteria
3
Heat-killed S-Strain
+ R-Strain
Mouse died
Some how the killed bacteria was able to
pass “something” to the R-strain
That Transformed it to become deadly
Avery’s Experiments (enzymes & bacteria)
Experiments
Results
Conclusion
Enzymes to break down
proteins, carbohydrates,
lipids, RNA, & finally DNA
Deadly in spite of all
enzymes except one
that broke apart DNA
DNA is the transforming molecule that
made Griffith’s R-Strain bacteria turn
into an S-Strain bacteria
Hershey & Chase Experiments (bacteria & a virus called bacteriophage)
Experiments
Results
Conclusion
Radioactive Protein
Radioactive DNA
New phages = no radioactivity
New phages = radioactive
Protein does not make new phages
DNA makes new phages
DNA Structure:
1. Nucleosides
2. Nucleotides
3. Bases
Purines
Adenine, Guanine
Pyrimidines
Cytosine, Thymine
4. Chargaff’s Rule
5. Franklin/Wilkins
Watson/Crick
1 hexagon ring
+ 1 pentagon ring
Anti-Parallel 5’ – 3’
5’
3’
1 hexagon ring
A bonds to T
C bonds to G
3’
5’
Conclusion-DNA is a helical structure
With distinctive regularities.
nucleotide
PO4
How would your replicate
this DNA molecule?
N base
5 CH2
O
4
deoxyribose
3
OH
2
1
Which is the leading strand?
Lagging strand?
Which one has Okasaki Fragments?
What enzymes are involved?
What type of bonds are formed?
What is the end result?
Replication fork
DNA
polymerase I
DNA
polymerase III
lagging strand
Okazaki
fragments
5’
3’
primase
ligase
5’
SSB
3’
DNA
polymerase III
5’
3’
leading strand
direction of replication
3’
5’
helicase
Replication enzymes
 Helicase - unzips DNA
 single-stranded binding proteins - controls the unzipping of DNA




DNA polymerase III - main DNA building enzyme
Primase - lays down RNA primer on lagging strand
DNA polymerase I - editing, repair & primer removal **
Ligase - “glues” Okazaki fragments together on lagging strand
Telomeres
 Expendable,
non-coding sequences at
ends of DNA


short sequence of bases
repeated 1000s times
TTAGGG in humans
 Telomerase enzyme in certain
cells


enzyme extends telomeres
prevalent in cancers
 Ends of chromosomes are
eroded with each replication


an issue in aging?
telomeres protect the ends of
chromosomes
Genetic Material
Prokaryotic DNA
• Circular in shape
• In the cell’s cytoplasm
• Not wrapped around proteins
• Fewer average bp’s (base pairs)
• No introns
Eukaryotic DNA
• Linear in shape
• In the cell’s nucleus
• Wrapped around proteins
• More bp’s (base pairs)
• Introns and exons
What would happen if you put a eukaryotic DNA into a prokaryote?
Regulating Gene Expression
DNA is tightly wound around histone
proteins, making DNA inaccessible to
enzymes that would code for the genetic
information
Acetyl groups attach to the histones
Causing the tight compaction to unravel,
now allowing DNA to be susceptible to
activation (replication or transcription)
A methyl group (CH3) can be attached to a cytosine base on DNA, as
shown here. When a methyl group is attached to a base, the base
cannot be accessed to build nucleotides
Implications: What effect would that have on the gene’s expression?
Ribosomes
Prokaryotic ribosomes
• 70S (smaller)
• Synthesized and assembled
in the cytoplasm
• Simultaneous transcription
and translation
• Translation begins with f-met
• Sensitive to antibiotics
Eukaryotic ribosomes
• 80S (larger)
• Synthesized in the nucleolus
• Assembled in the cytoplasm
(free) or (attached) on the
Rough Endoplasmic Reticulum
• Transcription then translation
• Translation begins with met
Both
• Translation is powered by GTP (guanosine triphosphate)
• Terminate translation with a stop codon & release factor proteins
The “Central Dogma”
 flow of gene
 tic information within a cell
transcription
DNA
replication
RNA
translation
protein
DNA - RNA - Protein
All RNA’s (mRNA, rRNA, tRNA) are transcribed (made) in the nucleus
Transcription -
RNA
Where is 5’ and 3’?
the making of mRNA from a DNA template
Post-transcriptional processing
 Primary transcript

eukaryotic mRNA needs work after transcription
 Protect mRNA

from RNase enzymes in cytoplasm
 add 5' cap
mRNA
5' cap
PPP
 add polyA tail 5' GCH
 Edit out introns
3'
A
3
intron = noncoding (inbetween) sequence
eukaryotic
DNA
exon = coding (expressed) sequence
primary mRNA
transcript
mature mRNA
transcript
pre-mRNA
spliced mRNA
Transcription in Prokaryotes
 Initiation

RNA polymerase binds to promoter sequence on DNA
Role of promoter
1. Where to start reading
= starting point
2. Which strand to read
= template strand
3. Direction on DNA
= always reads DNA 3'5’
= transcribes DNA 5’3’
What do prokarotic mRNA lack in comparison to eukaryotic mRNA’s?
Ribosomes – made of rRNA and protein
 P site (peptidyl-tRNA site)

holds tRNA carrying growing
polypeptide chain
 A site (aminoacyl-tRNA site)

holds tRNA carrying next amino acid to
be added to chain
 E site (exit site)

empty tRNA
leaves ribosome
from exit site
tRNA structure
 “Clover leaf” structure
anticodon on “clover leaf” end
 amino acid attached on 3' end

Building a polypeptide
 Initiation

brings together mRNA,
ribosome subunits,
proteins & initiator tRNA
 Elongation
 Termination
RNA polymerase
DNA
Can you tell
the story?
amino
acids
exon
intron
tRNA
pre-mRNA
5' cap
mature mRNA
aminoacyl tRNA
synthetase
polyA tail
large subunit
polypeptide
ribosome
5'
small subunit
tRNA
E P A
codon
3'
Put it all
together…
Lactose digestion in E.coli begins with its hydrolysis by the enzyme ß-galactosidase. The gene
encoding ß-galactosidase, lacZ, is part of a coordinately regulated operon containing other genes
required for lactose utilization.
Which of the following figures correctly depicts the interactions at the lac operon when lactose is NOT
being utilized? (The legend below defines the shapes of the molecules illustrated in the options.)
Lac Operon
What’s it sound like it involves?
Lac = Lactose; Operon = Operates when it’s On
Which of the following figures correctly depicts the interactions at the lac operon when lactose is NOT being utilized?
Implications to Genetically modified plants:
a. Pest resistance?
b. Herbicide resistance?