Biochemistry Lecture 6. Functions of Nucleotides and Nucleic Acids Nucleotide Functions: –Energy...
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Transcript of Biochemistry Lecture 6. Functions of Nucleotides and Nucleic Acids Nucleotide Functions: –Energy...
![Page 1: Biochemistry Lecture 6. Functions of Nucleotides and Nucleic Acids Nucleotide Functions: –Energy for metabolism (ATP) –Enzyme cofactors (NAD + ) –Signal.](https://reader038.fdocuments.us/reader038/viewer/2022102818/56649e915503460f94b97462/html5/thumbnails/1.jpg)
Biochemistry
Lecture 6
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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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NucleotideNucleosideNucleobase
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Pyrimidine Nucleobases
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Purine Nucleobases
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UV Absorption of Nucleobases
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-D-ribofuranose in RNA
-2’-deoxy-D-ribofuranose in DNA
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N-Glycosidic Bond
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Polynucleotides
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Hydrolysis of RNA
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Hydrogen Bonding!
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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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Other forms of DNA
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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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Using DNA Structure
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Thermal Denaturation
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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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DNA Technologies
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DNA Cloning
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Restriction Enzymes
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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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PCR
PolymeraseChainReaction
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Site-Directed Mutagenesis
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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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DNA Fingerprinting
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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 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?
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DNA Microarrays: Design Two fundamental approaches• One-color array
– 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– Developed by Stanford University, 1996– Probes sometimes pipetted on the chip– Targets linked to either green or red fluorescent labels– Used often in academia
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