Amino Acids o Polar Non polar Aspartic Acid Alanine Glutamic Acid Glycine Arginine Lysine Valine Leucine Histidine Isoleucine Asparagine 0 Proline Glutamine 0 Phenylalanine Serine 0 Methionine Threonine 0 Tryptophan Tyrosine 0 Cytesine Makes short RNA primer complimentary to template in Replication Basic is positive acidic is negative o o Uncharged polar is more likely to interact with water Enzymes RNAaseH Primase Removes Rna primer o o o o o 2 sites elongation and repair endonuclease o DNA polymerase Reads short RNA Primer elongates ONLY WORKs 5 3 Reads old 3 5 Proofreads itself Does not use primer Reading rules are the same RNA polymerase Ligase Spliciesomes Joins together fragments in Okazaki or repair Finds 5 splice and 3 splice sites straddles intron snRNP comes in and are held together Breaks intron out Excised intron is degraded snRNPs recycled DOES NOT RELEASE THE RNA DURING o Histone acetylase Histone acetyl transferase Acetylates histone lysine o Histone deacetylase Deacetylates histone lysine Adds phosphate becomes negative Removes phosphate group Part of GAP GTPase Activating Protein Activates hydrolysis in G protein Used in DNA polymerase proofreading and base repair Assists protein in folding into correct conformation Kinase Phosphotase o GTPase Endonuclease Chaperones o Helicase o o o o o o o o Breaks the bond in DNA repair Excision nuclease Used in nucleotide excision repair Unzips genes Phosphodiesterase Topoisomerase Telomerase Reduces stress ahead of replication fork Uses reverse transcriptase Carries RNA template Elongates shortened fragments Controlled in adult cells active in germ stem GGGTTA Tests o DNA replication is semi conservative One strand is conserved as both o RNAi Double stranded RNA leads to silencing Comes in recognized by Dicer enzyme as foreign Snips into siRNAs pairs with dicer Matches to complimentary mRNA of interest removes rest of sequence No translation of areas no protein silencing Research silencing to study effects of gene knockdown dominant negative approach Polymerase chain reaction PCR o o Used to amplify specified DNA sequence o o Heating seperation breaks strand o Annealing cools down higher concentration of primers bind to DNA sample o o After 3 cycles you begin to get specific sequence of interest Take DNA primer mix with sample tac polymerase good for heat Elongation polymerase acts on primer and extends sequences o o Cycles continue to amplify that sequence Note Primer A should be able to bind to strand 2 B to strand 1 see drawing Gel Electrophoresis o Gel Ethidum Bromide EtBr agarose porous o Used to compare mix of DNA o o o Used to compare sequences similar sequences with compare with band stripes Sends electric charge which makes DNA move to terminal Larger molecules move slower stay closer to start on gel Nuclear Run On Assay o RNA polymerase DNA mRNA isolated during transcription freezing o mRNA tagged allowed to incubate and extend no new transcription o After use cDNA tag reverse transcriptase and run blot analysis to quantify o Allows comparison of mRNA transcription levels between cells tissues etc etc Southern Blotting o DNA analysis o Nitrocellulose membrane transfers DNA from gel o Detection Hybridization with radioactive P compliment to target DNA o o Master Gene o o o Interaction of certain proteins allow master gene to tell cells to differentiate Immature cell interacts with protein to become mature Test method transfect with plasmid Incubate membrane probe crowds around target DNA Intensity is the amount of DNA of interest Transfect with plasmid will have gene of interest and antibioctic You want a gene that is expressed solely in mature cell not both immature and mature discard hybrid s Stain with antigen to see expression is successful or not Signal Gene activation protein interaction differention mature development Yeast 2 Hybrid System Protein protein interaction How mutations affects protein interactions DNA is genetic material Radio labeled bacteriophages S marked protein P marked DNA Infected bacterial cells and isolated P marked bacteria Because P was incorporated scientists knew DNA carried genetic information Replication is bi directional Origin of replication will remain equidistant from the edge of DNA loops Protein Structure Protein Domains Help us to predict structure and function Similar linear sequences Aid in adding of complex functions Complex more domain variety Easily shuffled between proteins Alpha and Beta sheets Type of secondary structure Alpha helical Formed by hydrogen bonds hydrophilic polar peptide backbone hidden in middle Arranged with alpha carbon in the middle side chains out side Gap between so small nothing can fit through it Beta Primary Anti parallel or parallel This is the linear sequence Contains all the information for 3D folding Is organization into alpha and beta sheets Folding primarily non covalent bonds Spatial arrangement of a and beta as well as connecting Aas Determines shape and structure Assited by chaperone proteins Di Sulfide bond IS A COVALENT BOND b w two cysteines Joining together of multiple teritary structures Affect of heating Denatures protein to its linear chain Effects the non covalent bonds Secondary Tertiary Quaternary o Bond Types Covalent Non Covalent Share electrons Arent affected by heat Ionic Electrostatic interactions Partial dipoles Weakened water Hydrogen bonds o Cell Building Blocks DNA RNA Nucleic Acids DNA deoxyribose Sugar H is partial positive due to electronegativity Holds DNA strands together Van der Waals Happens but so weak just interactions cause temporary dipole 2 complimentary strands anti parallel double helix Purines Adenine Guanine Pyrimidine Cytosine Thymine Matches A T 2 hydrogen bonds C G 3 hydrogen Backbone Phosphate and sugar Joined by phosphodiester bond diphosphate removed and remaining 5 phosphate joined to 3 carbon condensation reaction RNA ribose Sugar RNA is very similar know that it has an extra oxygen RNA Uracil U replaces T and binds to A RNA can be single stranded and take on several conformations Information storage and such Protein Amino group bound to alpha carbon bound to caroxyl group R group characterized by amino acids Left terminus is amino group right hand is carboxl Each is joined through peptide bond condensation reactions o Oligosaccharides Sugars Monosaccharides Cell structure Energy storage Lipids Fatty acids Membranes Amphipathic Hydrophilic head and Hydrophobic tail Repair Mechanisms DNA uses