Structure of Informational Molecules: DNA and RNA Stryer Short course Chapter 33.
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Transcript of Structure of Informational Molecules: DNA and RNA Stryer Short course Chapter 33.
![Page 1: Structure of Informational Molecules: DNA and RNA Stryer Short course Chapter 33.](https://reader035.fdocuments.in/reader035/viewer/2022062314/56649eaa5503460f94baf690/html5/thumbnails/1.jpg)
Structure of Informational Molecules: DNA and RNA
Stryer Short courseChapter 33
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Nucleic Acid Structure
• Nucleobase• Nucleoside• Nucleotide• Nucleic acid• Chromatin• Chromosome
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Polymeric Structure
• Polymer ideal for informational molecule
• Ribose and deoxyribose
• Numbering system
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Directionality
• 5’ 3’ directionality by convention
• 3’ 5’ phosphodiester linkage
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Base Structure• Purines and pyrimidines• Aromatic• Tautomers
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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
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Nucleotides
• pA is normally called _______ or ____________
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Problem
• List 4 ways that ATP differs from 3’-dGMP.
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Polynucleotides
• Phosphate diesters• polyanion• Abbreviation is
pdApdGpdTpdC• Tetranucleotide• Oligonucleotide• Exonucleases and
endonucleases
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Double Helix
• B-DNA• Chargoff’s Rule• Antiparallel• Right handed twist
ladder
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Complementary Base Pairs
Mismatching may occur with tautomers
N
N
HN
N N
N
NH
N
O
H
H
H
H
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
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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
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Denaturation
• Melting point• Melting curve• UV-absorption• cooperative
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Problem
• 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.
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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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Helical Forms
• B- form is major• A-form is similar
to RNA/RNA and hybrid DNA/RNA structures
• Z-DNA not understood, but shows flexibility of structure
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Major/Minor Groove in B-DNA
• 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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Semiconservative Replication
• Meselson and Stahl• Density gradient equilibrium centrifugation
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Explain the Results
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Bacterial DNA• Closed, circular
DNA• Supercoiling• Topology and
topoisomerases
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Eukaryotic DNA
• Highly compacted (by factor of 104) into chromatin (DNA/protein complex)
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RNA Structure
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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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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
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Recombinant DNA Techniques
Optional Lecture
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DNA Sequencing• DNA Polymerase: 5’ 3’• Sanger method• dideoxynucleotides
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Pyrosequencing
• 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– Denature– Anneal primer– Polymerase– Repeat
• Taq polymerase• Exponential production
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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
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Endonuclease
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Restriction Enzyme• Endonucleases recognize palindromes• Sticky ends and blunt ends
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Problem
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
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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
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Site-directed Mutagenesis
• Point mutations• Examine
importance of a residue
• Modify protein function