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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 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 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 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 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. Double Helix • B-DNA • Chargoff’s Rule • Right-handed twist ladder Double Helix Structure • • • • Antiparallel Dimensions-10 bp/turn Major/minor grooves Sugar phosphate backbone toward solvent • Base pairs stacked, perpendicular Complementary Base Pairs Mismatching may occur with tautomers Major/Minor Groove • Many pictures show ladder with backbone at 180o • Actually a distorted ladder with poles closer to each other, on one side • Major/minor groove recognition Weak Forces Stabilize Double Helix • Stacking interactions (vdW forces) • Hydrophobic effect • Ionic interactions • Hydrogen bonding Thermodynamic Analysis • Which factors are favorable in the double helix? • Which factors are favorable for denaturation? Native State Pi Stacking Hydrophobic Effect H-bonding Ionic interactions Entropy of strands Denatured 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 Hbonds, 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 histonedepleted chromosome: DNA strands have fallen off of scaffold RNA Structure RNA/DNA hybrid tRNA 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 Biotechnology 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 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