Genes to Proteins Pratt & Cornely Chapter 3

Nucleic Acid Structure Nucleobase Nucleoside Nucleotide Nucleic acid Chromatin Chromosome

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 ____________

Other Functions Nucleotides are used as energy storage (ATP) or combined with vitamins to make cofactors (NAD +, NADP +, CoA)

Polynucleotides Phosphate diesters polyanion directionality 5’  3’ 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

Major/Minor Groove Many pictures show ladder with backbone at 180 o Actually a distorted ladder with poles closer to each other, on one side

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 19 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

Reannealing

Bacterial DNA Closed, circular DNA Supercoiling Topology and topoisomerases

Eukaryotic DNA

Chromosome Scaffold of RNA and protein 30nm fibers are looped many times Picture of histone- depleted chromosome: DNA strands have fallen off of scaffold

RNA Structure RNA/DNA hybrid tRNA

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

Central Dogma

Transcription RNA polymerase 5’ to 3’ growth mRNA matches coding strand Except mRNA contains U, not T

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

Translation Ribosome rRNA tRNA

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, PP i 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 62 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

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