Structure of Informational Molecules: DNA and RNA
Transcript of Structure of Informational Molecules: DNA and RNA
Structure of Informational Molecules: DNA and RNA
Stryer Short courseChapter 33
Nucleic Acid Structure
• Nucleobase• Nucleoside• Nucleotide• Nucleic acid• Chromatin• Chromosome
Polymeric Structure
• Polymer ideal for informational molecule
• Ribose and deoxyribose
• Numbering system
Directionality
• 5’ 3’ directionality by convention
• 3’ 5’ phosphodiester linkage
Base Structure• Purines and pyrimidines• Aromatic• Tautomers
Nucleosides• Ribonucleosides and deoxyribonucleoside• Purine = osine; pyrimidine = idine (watch cytosine)
Nucleotides
• Phosphorylated on 2’, 3’, or 5’
• 5’ unless noted• Letter abbreviations
• Draw these:– dA– ADP– ppAp
Nucleotides
• pA is normally called _______ or ____________
Problem
• List 4 ways that ATP differs from 3’‐dGMP.
Polynucleotides
• Phosphate diesters• polyanion• Abbreviation is pdApdGpdTpdC
• Tetranucleotide• Oligonucleotide• Exonucleases and endonucleases
Double Helix
• B‐DNA• Chargoff’s Rule• Antiparallel• Right handed twist ladder
Complementary Base Pairs
Mismatching may occur with tautomers
N
N
HN
N N
N
NH
N
O
H
H
H
H
Adenine tautomer Cytosine
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
Weak Forces Stabilize Double Helix
• Stacking interactions (vdW forces)
• Hydrophobic effect• Charge‐charge• Hydrogen bonding
– Little contribution to stability
– Large contribution to selectivity
Denaturation
• Melting point• Melting curve• UV‐absorption• cooperative
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.
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
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
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
Semiconservative Replication
• Meselson and Stahl• Density gradient equilibrium centrifugation
Explain the Results
Bacterial DNA• Closed, circular DNA
• Supercoiling• Topology and topoisomerases
Eukaryotic DNA
• Highly compacted (by factor of 104) into chromatin (DNA/protein complex)
RNA Structure
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
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
Recombinant DNA Techniques
Optional Lecture
DNA Sequencing• DNA Polymerase: 5’ 3’• Sanger method• dideoxynucleotides
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
Polymerase Chain Reaction
• PCR– 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
Catalytic Hydrolysis: Nucleases
• Enzymes can catalyze hydrolysis
• Very important reactions!
• Nucleases– RNase vs DNase
• Single/double strand– Exonuclease vsEndonuclease
– Orientation of hydrolysis
Endonuclease
Restriction Enzyme• Endonucleases recognize palindromes• Sticky ends and blunt ends
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.
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