Exam 2 Bacteria single chromosome some plasmids present circular or linear dsDNA DNA in cytosol no histones Archaea one chromosome some plasmids circular dsDNA DNA in cytosol histones Eukary 2 chromosomes plasmid in fungi protozoa linear dsDNA in nucleus circular dsDNA in mt chloro histones DNA structure 4 nucelotides A G C T backbone alternating phosphates and deoxyribose pentose sugar phosphate connect 3 C of one sugar to 5 C of next sugar double stranded DNA antiparallel w complementary sequences double helix A T C G Chargaff s Rule grows by adding to 3 hydroxyl OH 5 phosphate bonds to 3 OH grows 5 to 3 only only add to 3 end needs enzyme DNA polymerase dehydration synthesis b c H2O is broken when NT adds RNA A U G C ribose sugar has O on every carbon Plasmids extra pieces of DNA replicate separately from chromosome great majority are double stranded mostly circular generally beneficial for cell ie antibiotic resistance DNA replication requires template DNA DNA polymerase Primase nucleotides RNA replication fork bidirectional replication and replisome proofreading and termination replication is semiconservative each of 2 progeny have one parental and one new strand DNA polymerase can NOT initiate synthesis of polynucleotide only ADD nts to 3 end of existing strand initial nt strand is required primer made of RNA made by primase leading strand primed once at ori lagging strand primed at EACH Okazaki fragment 1 start at origin of replication 2 bubble opens and double strand splits 3 helicase unwinds DNA 4 primase lays down primer and goes 5 3 5 DNA polymerase III synthesizes leading strand 6 lagging strand synthesized 7 DNA polymerase I destroys primer and creates nicks 8 ligase repairs nicks consume 1 ATP nick it repairs antiparallel elongation leading strand v lagging strand leading strand goes towards the fork lagging strand goes away from the fork Prokaryotic DNA replications termination of replication Central Dogma DNA RNA Protein Transcription of DNA to RNA 1 Promoter 2 RNA Polymerase 3 Sigma factor a 35 sequnce 10 seq aka Pribnow box TATA box Operon one promoter drives coding of multiple different genes Mutations silent no change in amino acid seq missense slightly different amino acid seq nonsense polypeptide synthesis ceases frameshift insertion major difference in amino acid seq frameshift deletion major difference in amino acid seq Sources of Mutation mutations are changes to genotype mis incorporation of bases by DNAPolym radiation radicals UV light specific structural change makes thymine dimers Thymine dimers T s fuse together after UV exposure lethal if not repaired chemical mutagens base analogs 5 bromouracil T is replaced with bU bU pairs with G G paired with C in new strand instead of T pairing with A get missense or nonsense mutation nt altering chem aflatoxin GC to AT switch DNA intercalating chem get in b w base pairs Cause bubbles in DNA Make deletions and insertions to account for this intercalation Cells know which strand to repair b c after replication DANMethylase modifies bases in DNA by adding methyl groups to them repair UNmethylated strands b c this new strand is more likely to contain mistakes Mechanisms of repair mutations that occur during DNA rep repaired by proofreading by DNAPolym mutations not repaired by proofreading are repaired by mismatched repair and excision post rep mutations that occur spontaneously any time are repaired by excision base or nucleotide excision thymine dimer light repair light activated repair enzyme 1 2 T dimer dark repair dna polym1 and ligase repair gap 3 Base excision repair enzymes remove incorrect nt DNAPI and ligase repair 4 Mismatch repair enzyme removes incorrect segment DNP3 repairs gap Mutations happen in random manner some mutations happen to confer function Ames Test mix his salmonella cells w known mutation rate liver extract and chemical look at potential mutagens cancer causing become mutagens in body count his colonies compared to control F plasmid encode all protein that makes pilus for conjugation only F and F can mate F F cannot mate Recombination F plasmid integrate into donor chromosome become Hfr F Hfr transfer DNA to F how much DNA going through depends on time of mating donor dna and recipient dna recombine to make recombinant F cell stays F because vast majority of functions genes stay with donor chromosome E coli conjugation map tells you how long it takes for it to go into recipient cell 100 mins marks on map are various genes and names bacteriophage viruses for bacteria obligate parasites not much metabolic function when outside of cell bacterial genome kept in head tail that helps it interact with cell surface and inject genes into cell bacteriophage multiply inside microbes until pressure so high it lyses transduction transfer of genes from one bacterial cell to another by bacteriophage sometimes by mistake make transducing phage host dna goes into developing phage transducing dna injected to next host will recombine with host dna restriction endonucleases in bacteria are barriers to gene transfer enzymes that cleave dna at diff specific recognition seq don t cut cells own dna b c they are protected by methylation palindromic cuts make sticky ends or blunt ends cut phosphate backbone H bonds ex EcoRI BamHI restriction fragments from two different org cut by same restricition enzyme can recombine with ligase regulation of gene expression allows organism to adapt to changing environment allow org to conserve energy if not making transcribing translating not using ATP allow org to switch developmental growth process principles of gene regulation transcription constitutive some genes are on not regulated always on inducible off can be turned on when signal induces them ex lac operon repressible on can be shut off by signal ex tryptophan operon enzymes allosteric site binding with effector causes inhibition so substrate cannot bind rxn inhibited active site binding with substrate allows reaction to proceed translational control no enzyme synthesis make mRNA but mRNA never translated cells invest energy to make mRNA but signal for translation not there to save energy best place for bacterial cell to stop before producing proteins is before transcription this means mRNA is not made and mRNA not translated lac operon lacI regulatory gene cell always makes lacI at some low level constituitive if no lactose lacI binds to operator and transcription is stopped if lactose lacI and lactose bind and