DNA Replication and Gene Expression Cultivation Independent Methods Great Plate Count Anomaly microbes can be cultivated Enter metagenomics The biosphere is so large but only about 1 of o Allows us to look at an organism s DNA and gather info about how it behaves Bacteria replicate by binary fission Replication happens at its maximum log phase when nutrients are at their maximum Parent cell prepares for division by enlarging its cell wall membrane and volume Duplicated chromosomes move to opposite ends of the cell o But How Bacteria do not have cytoskeletal fibers as eukarya do to separate chromosome so how do they know where to go o Theories Replisome1 or polymerase pushes daughter chromosomes to opposite ends of the cell Condensation of daughter chromosome pulls DNA to each end MreB murein cluster B Cytoskeletal protein similar to eukaryotic actin which provides structure Forms spiral inside periphery Experiments have shown that if MreB is mutated chromosomes do not segregate Cytoplasmic components are distributed to the two developing cells Septation occurs o The formation of cross walls between daughter cells o Several steps Assembly of Z Ring Critical Step Z ring splits cytoplasm in two Protein FtsZ o Tubulin homologue found in most bacteria and archaea o Forms the Z ring MinCDE system in E coli o Limits the Z ring to center of the cell o Min C Min D Min E oscillate from one side of the cell to the other which prevents Z ring formation at poles o The Z ring can only form in an area that lacks Min CDE 1 Replisome group of proteins that carry out replication DNA synthesis Z ring links to plasma membrane Constriction of cell and septum formation Daughter cells are divided after septation Some species will separate but others will o Smooth pathogenic surrounded by a huge capusule which is encoded for in the plasmid This plasmid is what allows it to cause disease and is not easily remain linked Griffith Experiment 2 types of streptococcus pneumonia removed from the cell o Rough non pathogenic Inject rats with o Killed S bacteria mouse lives o Live S bacteria mouse dies o Live R bacteria mouse lives o Killed S and Live R mouse dies Avery MacLeod McCarty Experiment Attempt to discover what was responsible for Griffith s transformation effect DNA RNA or protein Degraded each of these components one at a time Mixed R cells with the suspected transforming factor from heat killed S cells and added o Protease S colonies form o RNase S colonies form o DNase No colonies form Concludes that DNA is the transforming principle and directs the phenotype of cells Hershey Chase Experiment Used a T2 virus to infect E coli Procedure to label proteins In a viral infection for bacteriophages only the genes of a virus enter a host cell The capsid stays outside o Grew the T2 bacteriophages in the presence of either 32P to label DNA or 35S o Infect bacteria with the radioactive phages o Agitate the culture blend them o Centrifuge solutions o Viral capsids will be found in supernatant viral genes inside cells will be found in the pellet proteins Radioactive protein in supernatant and radioactive DNA in pellet Therefore viral genes consist of DNA and the coat consists of DNA Structure Double stranded and double helix Primary Structure o Nucleotide sequence A T 2 H bonds G C 3 H bonds Always a double ring bonded to a single ring Organisms in high temperatures will want more G C bonds in their DNA because they are stronger and it will keep them from melting In cold temperatures more A T bonds are useful because they are not as tightly held and so they will be less likely to fracture and break o Sugar Phosphate Backbone 5 carbon deoxyribose sugar linked to a phosphate group by a strong phosphodiester bond o Directionality is ALWAYS 5 3 Secondary structure o Antiparallel run in opposite directions to allow the bases to line up and One strand runs 5 3 and one strand runs 3 5 but always build in hydrogen bond together the 5 3 direction o Double helix o Complementary base pairing A T G C Bacteria Archaea o Singular circular chromosome o Single circular chromosome packaged around histone proteins2 Replication in Bacteria Singular origin of replication OriC o 245 BP long Contain many A T bonds because they are weaker and easier to break Begins with o 3 13 base pair repeats o Series of 4 9 BP repeats DnaA boxes DnaA proteins come to bind to the DnaA boxes o Physically folds and unwinds that region o Essential step After DnaA binds DnaB helicase gets delivered by DnaC unwinds the strand directionally SSB proteins single strand binding proteins keep the strand open 2 Histone proteins have a positive charge and congregate around the negatively charged DNA molecule to allow for tighter compaction Bacteria do not have histone proteins because they don t need that much compaction and it takes a lot of energy to make those proteins Bacteria generally have a linker protein in the center of its circular chromosome and DNA will loop around it coiling and supercoiling This is the most compaction observed in bacteria DnaG primase comes in and lays the initial RNA primers down DNA polymerase III starts replicating o Needs the help of DnaN a sliding clamp DNA polymerase I excises the RNA primer and fills in the gaps o Major proofreading enzyme o Is encoded by polA o DnaQ is the subunit responsible for the proofreading activity DNA ligase comes in and links the nucleotides together Termination occurs at the ter site o Directly opposite the oriC therefore singular termination site o Tus proteins bind the ter site stops replication o DNA molecules will be linked after termination Topoisomerase clips the backbones of DNA to separate them then relinks the backbones individually to yield 2 separate molecules FtsZ may also aid in separation Transcription in Bacteria genes mRNA Gene a segment of DNA that gets transcribed into ssRNA Main differences between eukaryotic and prokaryotic gene expression come in here 3 stages initiation elongation termination Initiation o Begins at the promoter Contains 2 defined consensus sequences at 10 and 35 10 TATA box TATAAT 35 TTGACA not quite as many A T bonds several G C o RNA polymerase a holoenzyme binds to the promoter Specifically the sigma factor recognizes the promoter3 RNA polymerase is composed of a core enzyme composed of polypeptides B B w and 2 copies of alpha and the sigma factor which combines with the core enzyme weakly o Once the holoenzyme binds to the promoter sigma factor