structure of informational molecules: dna and rna
TRANSCRIPT
![Page 1: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/1.jpg)
Structure of Informational Molecules: DNA and RNA
Stryer Short courseChapter 33
![Page 2: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/2.jpg)
Nucleic Acid Structure
• Nucleobase• Nucleoside• Nucleotide• Nucleic acid• Chromatin• Chromosome
![Page 3: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/3.jpg)
Polymeric Structure
• Polymer ideal for informational molecule
• Ribose and deoxyribose
• Numbering system
![Page 4: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/4.jpg)
Directionality
• 5’ 3’ directionality by convention
• 3’ 5’ phosphodiester linkage
![Page 5: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/5.jpg)
Base Structure• Purines and pyrimidines• Aromatic• Tautomers
![Page 6: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/6.jpg)
Nucleosides• Ribonucleosides and deoxyribonucleoside• Purine = osine; pyrimidine = idine (watch cytosine)
![Page 7: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/7.jpg)
Nucleotides
• Phosphorylated on 2’, 3’, or 5’
• 5’ unless noted• Letter abbreviations
• Draw these:– dA– ADP– ppAp
![Page 8: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/8.jpg)
Nucleotides
• pA is normally called _______ or ____________
![Page 9: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/9.jpg)
Problem
• List 4 ways that ATP differs from 3’‐dGMP.
![Page 10: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/10.jpg)
![Page 11: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/11.jpg)
Polynucleotides
• Phosphate diesters• polyanion• Abbreviation is pdApdGpdTpdC
• Tetranucleotide• Oligonucleotide• Exonucleases and endonucleases
![Page 12: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/12.jpg)
Double Helix
• B‐DNA• Chargoff’s Rule• Antiparallel• Right handed twist ladder
![Page 13: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/13.jpg)
Complementary Base Pairs
Mismatching may occur with tautomers
N
N
HN
N N
N
NH
N
O
H
H
H
H
Adenine tautomer Cytosine
![Page 14: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/14.jpg)
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
![Page 15: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/15.jpg)
Weak Forces Stabilize Double Helix
• Stacking interactions (vdW forces)
• Hydrophobic effect• Charge‐charge• Hydrogen bonding
– Little contribution to stability
– Large contribution to selectivity
![Page 16: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/16.jpg)
Denaturation
• Melting point• Melting curve• UV‐absorption• cooperative
![Page 17: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/17.jpg)
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.
![Page 18: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/18.jpg)
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
![Page 19: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/19.jpg)
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
![Page 20: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/20.jpg)
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
![Page 21: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/21.jpg)
Semiconservative Replication
• Meselson and Stahl• Density gradient equilibrium centrifugation
![Page 22: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/22.jpg)
Explain the Results
![Page 23: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/23.jpg)
Bacterial DNA• Closed, circular DNA
• Supercoiling• Topology and topoisomerases
![Page 24: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/24.jpg)
Eukaryotic DNA
• Highly compacted (by factor of 104) into chromatin (DNA/protein complex)
![Page 25: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/25.jpg)
RNA Structure
![Page 26: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/26.jpg)
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
![Page 27: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/27.jpg)
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
![Page 28: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/28.jpg)
Recombinant DNA Techniques
Optional Lecture
![Page 29: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/29.jpg)
DNA Sequencing• DNA Polymerase: 5’ 3’• Sanger method• dideoxynucleotides
![Page 30: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/30.jpg)
![Page 31: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/31.jpg)
![Page 32: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/32.jpg)
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
![Page 33: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/33.jpg)
Polymerase Chain Reaction
• PCR– Denature– Anneal primer– Polymerase– Repeat
• Taq polymerase• Exponential production
![Page 34: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/34.jpg)
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
![Page 35: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/35.jpg)
Catalytic Hydrolysis: Nucleases
• Enzymes can catalyze hydrolysis
• Very important reactions!
• Nucleases– RNase vs DNase
• Single/double strand– Exonuclease vsEndonuclease
– Orientation of hydrolysis
![Page 36: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/36.jpg)
Endonuclease
![Page 37: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/37.jpg)
Restriction Enzyme• Endonucleases recognize palindromes• Sticky ends and blunt ends
![Page 38: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/38.jpg)
ProblemRestriction 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.
![Page 39: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/39.jpg)
Making a Cloning Vector
![Page 40: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/40.jpg)
Making a Cloning Vector
• ampR is gene for ampicillin resistance
• LacZ encodes galactosidase
![Page 41: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/41.jpg)
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
![Page 42: Structure of Informational Molecules: DNA and RNA](https://reader031.vdocuments.site/reader031/viewer/2022020411/586b65221a28ab3f0a8b5f97/html5/thumbnails/42.jpg)
Site‐directed Mutagenesis
• Point mutations• Examine importance of a residue
• Modify protein function