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 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