biochemistry lecture 4. pyrimidine nucleobases
TRANSCRIPT
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Biochemistry
Lecture 4
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Pyrimidine Nucleobases
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Purine Nucleobases
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Polynucleotides
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Hydrolysis of RNA
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Functions ofNucleotides and Nucleic
Acids• Nucleotide Functions:
– Energy for metabolism (ATP)– Enzyme cofactors (NAD+)– Signal transduction (cAMP)
• Nucleic Acid Functions: – Storage of genetic info (DNA)– Transmission of genetic info (mRNA)– Processing of genetic information (ribozymes)– Protein synthesis (tRNA and rRNA)
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Discovery of DNA Structure
• One of the most important discoveries in biology
• Why is this important– "This structure has novel features which are of
considerable biological interest“--- Watson and Crick, Nature, 1953
• Good illustration of science in action:– Missteps in the path to a discovery– Value of knowledge– Value of collaboration– Cost of sharing your data too early
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Covalent Structure of DNA (1868-1935)
• Friedrich Miescher isolates “nuclein” from cell nuclei
• Hydrolysis of nuclein:– phosphate– pentose– and a nucleobase
• Chemical analysis:– phosphodiester linkages– pentose is ribofuranoside
O
OH
H HH
Thymine
H
CH2O P
OH
O
O
P OOH
OH
O
H
H HH
Adenine
H
CH2O P
OH
O
O
Structure of DNA: 1929
(Levene and London)
Structure of DNA:
1935(Levene and Tipson)
C5H7OThymine
O
P
O
POH
OH
O
O
O
OH
C5H7OAdenine
O
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Road to the Double Helix• Franklin and Wilkins:
– “Cross” means helix– “Diamonds” mean
that the phosphate-
sugar backbone
is outside– Calculated helical
parameters
• Watson and Crick:
– Missing layer means
alternating pattern
(major & minor groove)
– Hydrogen bonding:
A pairs with T
G pairs with C
Double helix fits the data!
Watson, Crick, and Wilkins shared 1962 Nobel Prize
Franklin died in 1958
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Hydrogen Bonding!
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The Central Dogma
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DNA Replication
“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material”
Watson and Crick, in their Nature paper,1953
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Other forms of DNA
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Using DNA Structure
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Why detect Transcription Factor targets?
• Transcription factors are medically relevant– ~10% of human
genes– Crucial roles in
development and cell life cycle
– Misregulation and mutation cause disease
– Critically, most cancers involve TF overactivity
Darnell, Nature Reviews Cancer 2, 740 (2002)
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Traditional methods for Transcription Factor
detectionExpression Microarrays Gel Shift Assays
The challenge: Most of these methods are indirect, slow (hours),
or can’t differentiate active and inactive protein.
Western Blots
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Bio-mimicry is a powerful motivation
Velcro: inspired by burrs Conformation Switching Probes
Marvin J S et al. PNAS 1997;94:4366-4371
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Optical Conformation SwitchingTF Switch Sensors
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Rationally Tuning TF Sensors
% s
witc
he
s o
pe
n
KS = 10
KS = 1
KS = 0.1 KS
= 0
.01
KS
= 0
.001
KS = [ ]
[ ]
KS [target]
KD (1+ KS) + KS [target] % switches open =
KD = [ ]
[ ]
[ ]
Target [M]
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TF Beacon Actual Performance
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Quantitative Detection in 4 easy steps
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Molecular Mechanisms of Spontaneous Mutagenesis
• Deamination• Very slow reactions• Large number of residues• The net effect is significant: 100 C U
events /day in a mammalian cell
• Depurination• N-glycosidic bond is hydrolyzed• Significant for purines: 10,000 purines
lost/day in a mammalian cell
• Cells have mechanisms to correct most of these modifications.
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UV Absorption of Nucleobases
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Pyrimidine Dimers from UV
http://highered.mcgraw-hill.com/olc/dl/120082/micro18.swf
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DNA Technologies
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DNA Cloning
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Restriction Enzymes
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PCR
PolymeraseChainReaction
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Antibiotic Selection• Antibiotics, such as penicillin and ampicillin, kill
bacteria
• Plasmids can carry genes that give host bacterium a resistance against antibiotics
• Allows growth (selection) of bacteria that have taken up the plasmid
NH
S
N
N
OO
H
OOH
HH
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Expression of Cloned Genes
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Protein Purification
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Eukaryotic Gene Expression in Bacteria
• An eukaryotic gene from the eukaryotic genome will not express correctly in the bacterium
• Eukaryotic genes have– Exons: coding regions– Introns: noncoding regions
• Introns in eukaryouric gene pose problems• Bacteria cannot splice introns out• mRNA is intron-free genetic material
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cDNA
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DNA Electrophoresis
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DNA Sequencing
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DNA Sequencing
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Shotgun Sequencing
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Electrochemical Sequencing
http://www.youtube.com/watch?v=yVf2295JqUg
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