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Genes to Proteins
Pratt & Cornely
Chapter 3
Nucleic Acid Structure
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Nucleobase
Nucleoside
Nucleotide
Nucleic acid
Chromatin
Chromosome
Base Structure
• Purines and pyrimidines
• Aromatic
• Tautomers
Nucleosides
• Ribonucleosides and deoxyribonucleoside
• Purine = osine; pyrimidine = idine
Nucleotides
• Phosphorylated
on 2’, 3’, or 5’
• 5’ unless noted
• Letter
abbreviations
• Draw these:
– dA
– ADP
– ppAp
Nucleotides
• pA is normally called _______ or ____________
Other Functions
• Nucleotides are used as energy storage (ATP) or
combined with vitamins to make cofactors (NAD+,
NADP+, CoA)
Polynucleotides
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Phosphate diesters
polyanion
directionality
5’  3’
Abbreviation is
pdApdGpdTpdC
• Tetranucleotide
• Oligonucleotide
• Exonucleases and
endonucleases
Catalytic Hydrolysis: Nucleases
• Enzymes can catalyze
hydrolysis
• Very important
reactions!
• Nucleases
– RNase vs DNase
• Single/double strand
– Exonuclease vs
Endonuclease
– Orientation of
hydrolysis
Endonuclease
Restriction Enzyme
• Endonucleases recognize palindromes
• Sticky ends and blunt ends
RNA Structure, Stability, and Function
• Structural difference of
2’ hydroxyl
– H-bonding in RNA
structure
– Reactions of catalytic
RNA (rare)
– Hydrolysis
• Structure dictates role
difference in DNA/RNA
Problem 62
Restriction enzymes are used to
construct restriction maps of DNA.
These are diagrams of specific DNA
molecules that show the sites
where the restriction enzymes
cleave the DNA. To construct a
restriction map, purified samples of
DNA are treated with restriction
enzymes, either alone or in
combination, and then the reaction
products are separated by agarose
gel electrophoresis. Use the results
of this gel to construct a restriction
map for this sample of DNA.
Double Helix
• B-DNA
• Chargoff’s Rule
• Right-handed twist
ladder
Double Helix Structure
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Antiparallel
Dimensions-10 bp/turn
Major/minor grooves
Sugar phosphate
backbone toward
solvent
• Base pairs stacked,
perpendicular
Complementary Base Pairs
Mismatching may occur with tautomers
Major/Minor Groove
• Many pictures
show ladder with
backbone at 180o
• Actually a
distorted ladder
with poles closer
to each other, on
one side
• Major/minor
groove
recognition
Weak Forces Stabilize Double Helix
• Stacking interactions
(vdW forces)
• Hydrophobic effect
• Ionic interactions
• Hydrogen bonding
Thermodynamic Analysis
• Which factors are
favorable in the double
helix?
• Which factors are
favorable for
denaturation?
Native State
Pi Stacking
Hydrophobic Effect
H-bonding
Ionic interactions
Entropy of strands
Denatured
Denaturation
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Melting point
Melting curve
UV-absorption
cooperative
Problem 19
• True or False: Because a G:C base pair is
stabilized by three hydrogen bonds, whereas
an A:T base pair is stabilized by only two
hydrogen bonds, GC rich DNA is harder to
melt than AT-rich DNA.
A/T Rich and G/C Rich strands
• GC rich strands harder to denature due to
STACKING (not H-bonds)
• Cooperativity due to initial unstacking, which
exposes bases to water, which destabilizes Hbonds, which leads to further denaturation
Reannealing
Bacterial DNA
• Closed, circular DNA
• Supercoiling
• Topology and topoisomerases
Eukaryotic DNA
Chromosome
• Scaffold of RNA and
protein
• 30nm fibers are looped
many times
• Picture of histonedepleted chromosome:
DNA strands have fallen
off of scaffold
RNA Structure
RNA/DNA hybrid
tRNA
Central Dogma
Transcription
• RNA polymerase
• 5’ to 3’ growth
• mRNA matches coding
strand
• Except mRNA contains
U, not T
Why does DNA not contain U?
• DNA damage from UV
light, hydrolysis,
oxidation
• If DNA contained U, it
would be unable to
recognize a hydrolyzed
cytosine
• In RNA, damage not as
important, and T
production is costly
Translation
• Ribosome
• rRNA
• tRNA
Biotechnology
Optional Lecture
DNA Sequencing
• DNA Polymerase: 5’  3’
• Sanger method
• dideoxynucleotides
Pyrosequencing
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Attach DNA to a solid surface
Run dNTPs over DNA one at a time
If reaction occurs, PPi is produced
Linked to a luciferase
Light detected
Polymerase Chain Reaction
• PCR
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Denature
Anneal primer
Polymerase
Repeat
• Taq polymerase
• Exponential production
Recombinant DNA technology
• Recombinant DNA
– Allows incorporation of gene(s) into other DNA
– Cut with exonucleases, anneal, and ligate
• Recombinant DNA serves as a cloning vector
– Incorporate into cells
– Select cells that have been transformed
Making a Cloning Vector
Making a Cloning Vector
• ampR is gene for
ampicillin
resistance
• LacZ encodes
galactosidase
Selecting Transformed Bacteria
• Some plasmids are
recombinant, and some are
not
• Some cells accept a
plasmid, some accept
recombinant plasmid, and
some don’t accept any
• Transformed cells selected
by growing on a petri dish
with ampicilin and
galactose derivative
• Explain
Site-directed Mutagenesis
• Point mutations
• Examine
importance of a
residue
• Modify protein
function