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 chromosomeBacterial genetic elementsPlasmidsDNA replicationDNA replicationTHE central dogmaThe Central Dogma (1957)TRANSCRIPTIONRNA - Information CarrierInitiation of TranscriptionElongation of TranscriptionSlide 22TranslationTranslation elementsTransfer RNA (tRNA)Amino AcidsAmino Acids and Peptide BondsSlide 28Translation initiationElongation P, A, and E sites on ribosomeTerminationSlide 32Summary – cellular functionsWhen things go wrong…How mutations are madeSlide 36TransformationTransductionConjugationCELLULAR FUNCTIONS AND MICROBIAL GENETICSCELLULAR FUNCTIONS AND MICROBIAL GENETICSMIC 301Terry D. Connell, Ph.D.GeneticsGenetics•Gregor Mendel (1856-63)★“Father of modern genetics”, Austrian monk★Cultivating sweet pea plants★Discovered that ¼ expressed purebred recessive features, ½ were hybrids and ¼ had purebred dominant features. ★Two laws of InheritanceALLELES: Alternative forms of a geneI. Allele pairs separate independently during the formation of gametes. Thus, traits are transmitted to offspring independently of one another.II. Allele pairs separate or segregate during gamete formation and randomly unite at fertilizationRelationship Genes and ProteinsRelationship Genes and Proteins•Dr. Archibald Garrod (1909) ★Suggested a link between a human inherited disease (alkaptonuria) and the production of a specific protein enzyme.★Lacks enzyme for converting alkapton Oxygenized form of homogentisic acid★Lack of that enzyme results in the buildup of homogentisic acid (Dark red color in urine)★Genes and proteins are ‘linked’ in some fashion★Still, how hereditary information was stored was unknownDNA carries genetic informationDNA carries genetic information•Frederick Griffith (1928)•Oswald Avery (1944), MacLeod, 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 FOUR bases: Adenine, Cytosine, Thymine, Guanine★Polynucleotide that is organized into ‘chromosomes’Basespurines: 2 rings (adenine, guanine)pYrimidines: 1 ring (thYmine, cYtosine)DNA structure (1)DNA structure (1)•Erwin Chargaff (1950)★Showed that: Amounts of A + T bases = Amounts of C + G bases.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)★Using X-ray diffraction of DNA crystals: Double helix★Essential data 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 organisms•5’ phosphate (HPO3) and a 3’ hydroxyl (OH)Structure 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 DNAB form most common in living organismsA found under certain circumstancesZ found in high salt and polyanionsBacterial genetic elementsBacterial genetic elements•Chromosomes★Most often one dsDNA molecule★Vibrio cholerae: TWO!★Usually circular★0.5 to 10 million base pairs•Plasmids Extra-chromosomal DNA = autonomously replicate★A species can have several plasmids★Some species have no plasmids★Usually circular (Borrelia spp. – linear plasmids)★300 bp to 1.5 million base pairs★Copy number between 1 and 1,000Bacterial chromosomeBacterial chromosome•Usually circular•1-5 origins or replication (OriV)•Organized into a nucleoidNucleoid structures (lighter areas) bylight microscopy(Bohrmann et al 1991)Nucleoid 3D reconstitution from electron microscopy sections(Bohrmann et al 1991)Bacterial genetic elementsBacterial genetic elements•Chromosomes★Most often one dsDNA molecule★Vibrio cholerae: TWO!★Usually circular★0.5 to 10 million base pairs•Plasmids Extra-chromosomal DNA = autonomously(independently) (usually not always) replicate★A species can have several plasmids★Some species have no plasmids★Usually circular (Borrelia spp. – linear plasmids)★300 bp to 1.5 million base pairs★Copy number between 1 and 1,000PlasmidsPlasmids•Different types★Resistance – carrying genes for antibiotic resistance★Toxins – carrying toxin genes★Conjugation – carrying information for conjugation•Not usually required for survival of the bacterial 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 of the double-stranded DNA followed each daughter cell.•Mechanism:★Binding of DnaA to OriV (Origin of replication)★Helicase: Unwinds the helix★Formation of replication fork★SSB proteins hold single strand of DNA open★DNA Primase and RNA polymerase primes the reaction★DNA polymerase (Kornberg, 1959) adds new base pairs ★Polymerization occurs in one direction: 5’ > 3’★Leading strand – Continuous replication★Lagging strand – Okazaki fragments prime–Gaps sealed with DNA ligase★Termination at specific termination sequencesTHE CENTRAL DOGMATHE CENTRAL