Structure of Informational Molecules: DNA and RNA Stryer Short course Chapter 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

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 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 vs Endonuclease Orientation of hydrolysis

Endonuclease

Restriction Enzyme Endonucleases recognize palindromes Sticky ends and blunt ends

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.

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