Cellular functions and Microbial GeneticsGeneticsRelationship Genes and ProteinsDNA carries genetic informationWhat is DNA?DNA structure (1)DNA structure (2)DNA structure (3)DNA structureBacterial genetic elementsBacterial chromosomePlasmidsDNA replicationDNA replicationTHE central dogmaThe central dogma (1957)TRANSCRIPTIONRNA information CarrierInitiationElongationTerminationTranslationTranslation elementsCodon usageTransfer RNAAmino AcidsAmino Acids and Peptide BondsTranslation initiationElongationTerminationSummary – cellular functionsWhen things go wrong…How mutations are madeBacterial DNA transferTransformationTransductionConjugationCELLULAR FUNCTIONS AND MICROBIAL GENETICSCELLULAR FUNCTIONS AND MICROBIAL GENETICSMIC 301Anders Håkansson, Ph.D.January 23, 2013GeneticsGenetics•Gregor Mendel (1856-63)★“Father of modern genetics”, Austrian monk★Cultivating sweet pea plants, he discovered that ¼ expressed purebred recessive features, ½ were hybrids and ¼ had purebred dominant features. ★Work led to Medel’s two laws of inheritanceRelationship Genes and ProteinsRelationship Genes and Proteins•Dr. Archibald Garrod (1909) ★He suggested a link between a human inherited disease (alkaptonuria) and the production of a specific protein enzyme.★Lack of that enzyme results in the buildup of homogentisic acid which is excreted in the urine and gives it a dark red color.★Still, how hereditary information was stored was unknown.DNA carries genetic informationDNA carries genetic information•Frederick Griffith (1928)•Oswald Avery (1944) with MacLeod and McCarthy★Repeated Griffith’s experiment but added extract from bacteria instead of killed, whole bacteria.★Treated extract with protease, RNAse and DNAse. Only DNAse treatment inhibited transformation = DNA carried the genetic information.What is DNA?What is DNA?•Deoxyribonucleic acid (identified in the end of 1800s)★Consist of 4 bases★Polynucleotide, organized into chromosomesBasespurines: 2 rings (adenine, guanine)pYrimidines: 1 ring (thYmine, cYtosine)DNA structure (1)DNA structure (1)•Erwin Chargaff (1950)★Showed that the amount of bases A and T were always the same in DNA as were the amounts of C and G.Base pairing AT - 2 hydrogen bonds GC - 3 hydrogen bonds (stronger bond)Chargoff: A=T, G=C Base pairing AT - 2 hydrogen bonds GC - 3 hydrogen bonds (stronger bond)Chargoff: A=T, G=CDNA structure (2)DNA structure (2)•Maurice Wilkins and Rosalind Franklin (Early 1950s)★Provided the data, using X-ray diffraction, necessary for Watson and Crick to elucidate the structure of DNA.Photo 51 by Franklin, showing the B form of DNADNA structure (3)DNA structure (3)•James Watson and Francis Crick (1953)original papersNATURE|VOL 421|23 JANUARY 2003|www.nature.com/nature397© 2003 Nature PublishingGrouporiginal papersNATURE|VOL 421|23 JANUARY 2003|www.nature.com/nature397© 2003 Nature PublishingGrouporiginal papersNATURE|VOL 421|23 JANUARY 2003|www.nature.com/nature397© 2003 Nature PublishingGrouporiginal papersNATURE|VOL 421|23 JANUARY 2003|www.nature.com/nature397© 2003 Nature PublishingGroupDNA structureDNA structure•Deoxyribonucleic acid forms found in living organismsStructure of DNA• Double helix• 4 bases in pairs– A = Adenine– T = Thymine– G = Guanine– C = CytosineWatson & Crick 1953G-CA-Tcomplementary basesA B ZTop-viewSide-viewForms of DNABacterial genetic elementsBacterial genetic elements•Chromosomes★Most often one (more than one has been observed)★Usually circular★Between 0.5 and 10 million base pairs long•Plasmids (extra-chromosomal DNA = replicate by themselves)★A species can have several plasmids and some species have none★Usually circular★Between 1 thousand and 1.5 million base pairs★Copy number between 1 and 1,000Bacterial chromosomeBacterial chromosome•Usually circular with 1-5 origins or replication•Organized in a nucleoidNucleoid structures (lighter areas) bylight microscopy(Bohrmann et al 1991)Nucleoid 3D reconstitution from electron microscopy sections(Bohrmann et al 1991)PlasmidsPlasmids•Different types★Resistance – carrying genes for antibiotic resistance★Toxins – carrying toxin genes★Conjugation – carrying information for conjugation•Not usually required for survival of cell•Replicates independently of chromosome•Used extensively in molecular biology★Recombinant protein expression★CloningDNA REPLICATIONDNA REPLICATIONHow is DNA copied?DNA Replication• Split helix• Copy by pairing bases• semi-conservative replicationWatson & Crick 1953DNA replicationDNA replication•Semi-conservative replication ★Meselson and Stahl (1958) showed that one strand followed each daughter cell.•Mechanism:★Binding of DnaA toreplication origin★Open the helix with helicase = formation of replication fork★SSB proteins hold single strand open★Primase and RNA polymerase primes★DNA polymerase adds new base pairs 5’ to 3’★Leading strand – continuous★Lagging strand – Okazaki fragments requiring sealing with DNA ligase★Termination at specific termination sequencesTHE CENTRAL DOGMATHE CENTRAL DOGMAHow does DNA give rise to proteins?The central dogma (1957)The central dogma (1957)★DNA carries genetic information★Through transcriptionthe information is copiedto RNA★Through translationthe information in RNAis translated into a poly-peptide chain or proteinThe “Central Dogma”genestring of amino acidsDNARNApolypeptidechainTranscriptionTranslationThe “Central Dogma”genestring of amino acidsDNARNApolypeptidechainTranscriptionTranslationThe “Central Dogma”genestring of amino acidsDNARNApolypeptidechainTranscriptionTranslationTRANSCRIPTIONTRANSCRIPTIONCopying the genetic information into messenger RNARNA information CarrierRNA information Carrier•Francois Jacob and Jacques Monod (1960)★Genetic information in the nucleus★Proteins are synthesized on ribosomes (ribosomal RNA) in the cytoplasm.★Proposed an alternative of non-specialized ribosomes that translate unstable RNAs called messenger RNAInitiationInitiation★Transcription factors bind topromoter region (-10 and -35 bpbefore the start of transcription).★Promoters important regulatorydomains★RNA polymerase is recruited with the help of sigma factors (inbacteria most often sigma-70)★DNA helix is unwound★The promoter is cleared★Transcription