Download Biochemistry

Biochemistry
Lecture 6
Functions of
Nucleotides 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)
Nucleoside
Nucleotide
Nucleobase
Pyrimidine Nucleobases
Purine Nucleobases
UV Absorption of Nucleobases
• -D-ribofuranose in RNA
• -2’-deoxy-D-ribofuranose in DNA
-N-Glycosidic Bond
Polynucleotides
Hydrolysis of RNA
Hydrogen Bonding!
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:
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Missteps in the path to a discovery
Value of knowledge
Value of collaboration
Cost of sharing your data too early
Covalent Structure of DNA (1868-1935)
OH
HO
P
• Friedrich Miescher
isolates “nuclein” from
cell nuclei
O
O
Thymine
C5H7O
O
HO
P
O
Structure of DNA:
1929
O
Adenine
• Hydrolysis of nuclein:
C5H7O
(Levene and London)
– phosphate
– pentose
– and a nucleobase
O
OH
HO P O
H
H
O
H
H
Thymine
H
• Chemical analysis:
OH
CH2O
P O
– phosphodiester linkages
– pentose is ribofuranoside
O
Structure of DNA:
1935
(Levene and Tipson)
H
H
O
H
H
Adenine
H
OH
CH2O P O
O
O
Road to the Double Helix
• Watson and Crick:
– Missing layer means
alternating pattern
(major & minor groove)
– Hydrogen bonding:
A pairs with T
G pairs with C
• Franklin and Wilkins:
–“Cross” means helix
–“Diamonds” mean
that the phosphatesugar backbone
is outside
– Calculated helical
parameters
Double helix fits the data!
Watson, Crick, and Wilkins shared
1962 Nobel Prize
Franklin died in 1958
Other forms of DNA
The Central Dogma
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
Using DNA Structure
Thermal Denaturation
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.
DNA Technologies
DNA Cloning
Restriction Enzymes
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
OH
O
O
N
S
H
O
N
H
H N
H
PCR
Polymerase
Chain
Reaction
Site-Directed
Mutagenesis
DNA Electrophoresis
DNA Sequencing
DNA Sequencing
Shotgun
Sequencing
DNA Fingerprinting
Expression of Cloned Genes
Protein Purification
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
cDNA
DNA Microarrays: Applications
DNA Microarrays allow simultaneous screening
of many thousands of genes: high-throughput
screening
• genome wide genotyping
– Which genes are present in this
individual?
• tissue-specific gene expression
– Which genes are used to make
proteins?
• mutational analysis
– Which genes have been mutated?
DNA Microarrays: Design
Two fundamental approaches
• One-color array
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Patented and commerialized by Affymetrix
Photolitographic synthesis of probe DNA on the chip
Targets are biotin labeled
Bound targets detected using streptavidin-fluorofore
complex
Widely used in industry
Two-color array
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Developed by Stanford University, 1996
Probes sometimes pipetted on the chip
Targets linked to either green or red fluorescent labels
Used often in academia