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Tutorial - DNA
Watson & Crick
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Griffith’s Experiments (Streptococcus pneumoniae)
Experiment Results Conclusion
1 R-Strain bacteria S-Strain bacteria
2 Heat-killed S-Strain
3 Heat-killed S-Strain+ R-Strain
Avery’s Experiments (enzymes & bacteria)
Nonvirulent strain of bacteria
Destroyed deadly bacteria
Some how the killed bacteria was able to pass “something” to the R-strain
That Transformed it to become deadly
Experiments Results Conclusion
Enzymes to break down proteins, carbohydrates, lipids, RNA, & finally 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 ProteinRadioactive DNA
Protein does not make new phagesDNA makes new phages
Mouse lived
Mouse lived
Mouse died
Deadly in spite of all enzymes except one that broke apart DNA
New phages = no radioactivity
Mouse died Virulent/deadly strain
New phages = radioactive
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Anti-Parallel 5’ – 3’
DNA Structure:
1. Nucleosides
2. Nucleotides
3. Bases 1 hexagon ring+ 1 pentagon ring
1 hexagon ring
4. Chargaff’s Rule
PurinesAdenine, Guanine
PyrimidinesCytosine, Thymine
5’
5’3’
3’
A bonds to TC bonds to G
5. Franklin/Wilkins Watson/Crick
Conclusion-DNA is a helical structure With distinctive regularities.
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OH
CH2
O
4
5
3 2
1
PO4
N base
deoxyribose
nucleotide
How would your replicatethis DNA molecule?
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?
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Replication fork
3’
5’
3’
5’
5’
3’
leading strand
Okazaki fragments
lagging strand
3’ 5’
DNA polymerase III
ligase
helicase
direction of replication
primase
DNA polymerase III
SSB
DNA polymerase I
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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
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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
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Genetic Material
Prokaryotic DNA Eukaryotic DNA
• Circular in shape• In the cell’s cytoplasm• Not wrapped around proteins• Fewer average bp’s (base pairs)• No introns
• 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?
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DNA is tightly wound around histone proteins, making DNA inaccessible to enzymes that would code for the geneticinformation
Acetyl groups attach to the histonesCausing the tight compaction to unravel, now allowing DNA to be susceptible to activation (replication or transcription)
Regulating Gene Expression
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?
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Ribosomes
Prokaryotic ribosomes Eukaryotic ribosomes
• 70S (smaller)• Synthesized and assembled in the cytoplasm• Simultaneous transcription and translation• Translation begins with f-met• Sensitive to antibiotics
• 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
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proteinRNA
The “Central Dogma”
DNAtranscription translation
replication
flow of gene tic information within a cell
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DNA - RNA - ProteinAll RNA’s (mRNA, rRNA, tRNA) are transcribed (made) in the nucleus
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Transcription - the making of mRNA from a DNA template
RNA
Where is 5’ and 3’?
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A A A A A3' poly-A tail
CH3
mRNA
5'
5' cap
3'
G PPP
Post-transcriptional processing Primary transcript
eukaryotic mRNA needs work after transcription
Protect mRNA from RNase enzymes in cytoplasm
add 5' cap add polyA tail
Edit out introns
eukaryoticDNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
primary mRNAtranscript
mature mRNAtranscript
pre-mRNA
spliced mRNA
50-250 A’s
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Role of promoter1. 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’
Transcription in Prokaryotes Initiation
RNA polymerase binds to promoter sequence on DNA
What do prokarotic mRNA lack in comparison to eukaryotic mRNA’s?
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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
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tRNA structure “Clover leaf” structure
anticodon on “clover leaf” end amino acid attached on 3' end
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Building a polypeptide Initiation
brings together mRNA, ribosome subunits, proteins & initiator tRNA
Elongation Termination
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Can you tell the story?
DNA
pre-mRNA
ribosome
tRNA
aminoacids
polypeptide
mature mRNA
5' cap
polyA tail
large subunit
small subunit
aminoacyl tRNAsynthetase
E P A
5'
3'
RNA polymerase
exonintron
tRNA
codon
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Put it all together…
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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.)
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Lac OperonWhat’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?
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Implications to Genetically modified plants: a. Pest resistance? b. Herbicide resistance?