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6/4/2015
Genes to Proteins
Pratt & Cornely
Chapter 3
Nucleic Acid Structure
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Nucleobase
Nucleoside
Nucleotide
Nucleic acid
Chromatin
Chromosome
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Base Structure
• Purines and pyrimidines
• Aromatic
• Tautomers
Nucleosides
• Ribonucleosides and deoxyribonucleoside
• Purine = osine; pyrimidine = idine (watch cytosine)
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Nucleotides
• Phosphorylated on 2’, 3’, or 5’
• 5’ unless noted
• Letter abbreviations
• Draw these:
– dA
– ADP
– ppAp
Nucleotides
• pA is normally called _______ or ____________
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Other Functions
• Nucleotides are used as energy storage (ATP) or combined with vitamins to make cofactors (NAD+, NADP+, CoA)
Polynucleotides
Phosphate diesters
polyanion
directionality
5’  3’
Abbreviation is pdApdGpdTpdC
• Tetranucleotide
• Oligonucleotide
• Exonucleases and endonucleases
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Double Helix
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B‐DNA
Chargoff’s Rule
Antiparallel
Right handed twist ladder
Complementary Base Pairs
Mismatching may occur with tautomers
H
H
H
N
N
HN
N
H
N
N
NH
N
O
Adenine tautomer
Cytosine
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Double Helix Structure
• Dimensions‐10 bp/turn
• Major/minor grooves
• Sugar phosphate backbone toward solvent
• Base pairs stacked, perpendicular
• Edges of bases exposed in grooves for recognition
Major/Minor Groove
• Many pictures show ladder with backbone at 180o
• Actually a distorted ladder with poles closer to each other, on one side
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Weak Forces Stabilize Double Helix
• Stacking interactions (vdW forces)
• Hydrophobic effect
• Charge‐charge
• Hydrogen bonding
– Little contribution to stability
– Large contribution to selectivity
Denaturation
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Melting point
Melting curve
UV‐absorption
cooperative
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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 H‐
bonds, which leads to further denaturation
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Reannealing
Bacterial DNA
• Closed, circular DNA
• Supercoiling
• Topology and topoisomerases
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Eukaryotic DNA
Chromosome
• Scaffold of RNA and protein
• 30nm fibers are looped many times
• Picture of histone‐
depleted chromosome: DNA strands have fallen off of scaffold
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RNA Structure
RNA/DNA hybrid
tRNA
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
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Central Dogma
Transcription
• RNA polymerase
• 5’ to 3’ growth
• mRNA matches coding strand
• Except mRNA contains U, not T
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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
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DNA Sequencing
• DNA Polymerase: 5’  3’
• Sanger method
• dideoxynucleotides
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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
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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
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Catalytic Hydrolysis: Nucleases
• Enzymes can catalyze hydrolysis
• Very important reactions!
• Nucleases
– RNase vs DNase
• Single/double strand
– Exonuclease vs
Endonuclease
– Orientation of hydrolysis
Endonuclease
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Restriction Enzyme
• Endonucleases recognize palindromes
• Sticky ends and blunt ends
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.
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Making a Cloning Vector Making a Cloning Vector • ampR is gene for ampicillin resistance
• LacZ encodes galactosidase
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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
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