Genes to Proteins Pratt & Cornely Chapter 3 Nucleobase Nucleoside Nucleotide Nucleic acid Chromatin Chromosome Nucleic Acid Structure 1
Base Structure Purines and pyrimidines Aromatic Tautomers Nucleosides Ribonucleosides and deoxyribonucleoside Purine = osine; pyrimidine = idine (watch cytosine) 2
Phosphorylated on 2, 3, or 5 5 unless noted Letter abbreviations Draw these: da ADP ppap Nucleotides Nucleotides pa is normally called or 3
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 4
Double Helix B DNA Chargoff s Rule Antiparallel Right handed twist ladder Complementary Base Pairs Mismatching may occur with tautomers H H N N H N HN N H N N NH O Adenine tautomer Cytosine 5
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 6
Weak Forces Stabilize Double Helix Stacking interactions (vdw forces) Hydrophobic effect Charge charge Hydrogen bonding Little contribution to stability Large contribution to selectivity Melting point Melting curve UV absorption cooperative Denaturation 7
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 8
Reannealing Bacterial DNA Closed, circular DNA Supercoiling Topology and topoisomerases 9
Eukaryotic DNA Chromosome Scaffold of RNA and protein 30nm fibers are looped many times Picture of histonedepleted chromosome: DNA strands have fallen off of scaffold 10
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 11
Central Dogma Transcription RNA polymerase 5 to 3 growth mrna matches coding strand Except mrna contains U, not T 12
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 Ribosome rrna trna Translation 13
DNA Sequencing DNA Polymerase: 5 3 Sanger method dideoxynucleotides 14
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 15
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 16
Catalytic Hydrolysis: Nucleases Enzymes can catalyze hydrolysis Very important reactions! Nucleases RNase vs DNase Single/double strand Exonuclease vs Endonuclease Orientation of hydrolysis Endonuclease 17
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. 18
Making a Cloning Vector Making a Cloning Vector ampr is gene for ampicillin resistance LacZ encodes galactosidase 19
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 20