Lecture 15 Two component regulatory systems o Sensor response regulator o Uses the transfer of phosphate Chemotaxis and two component regulation repellents and methyl groups o Che Y exist in two states phosphorylated and not phosphorylated when phosphorylated it goes into a tumble it is NOT moving up and down the concentration gradient anymore o Che Y is phosphorylated by Che A o Che W and MCP methyl accepting chemotaxis protein help phosphorylate Che A motion repellents o MCP phosphorylates Che A when it is bound to compounds that are negative for o In this figure MCP is binding a repellent which then phosphorylates Che A Che A then phosphorylates Che Y Che Y because it is pushed into a more phosphorylated state is then pushed into a tumble not moving in any direction o Che R transfers methyl groups to the MCP the effect of binding methyl groups to the MCP is the same as binding a repellent it then phosphorylates Che A etc Che R adds the methyl groups on no matter what the environmental conditions is o Che B also exist in two states phosphorylated and not phosphorylated also done by Che A Che B s job is to take the methyl groups off of MCP o Che Z take phosphate group off Che Y Chemotaxis and two component regulation bound attractant o When bound to an attractant you get less phosphorylated Che A and therefore less phosphorylated Che Y and the cell goes into a run starts moving in a particular direction o However because Che R is still in the system it still continuously methylates MCP which phosphorylates Che A which phosphorylates Che Y and Che B Che B removes the methyl groups which then allows MCP to bound the attractant and lessens the number of phosphorylation and the cell continues to run Translation Brief description look over on your own o tRNA s charged with amino acids the first tRNA in any amino acid sequence will be the one that pairs with the AUG codon of the mRNA ex methionine o The next tRNA is read the peptide bond is formed between the two Be able to read codon table Type of mutations o Substitutions mismatching of nucleotides or replacement of one base pair by another Silent mutation if change results in redundant codon as amino acid sequence in polypeptide is not changed Missense mutation if change results in codon for a different amino acid effect depends on location of different amino acid in polypeptide Nonsense mutation if codon for an amino acid is changed to a stop codon o Frameshift insertion addition of one or a few nucleotide pairs creates new sequence of codons missense and nonsense mutations o Frameshift deletion removal of one or a few nucleotide pairs creates new sequence of codons missense and nonsense mutations Consequences of mutation o Altered enzymatic function Kinetics how fast it catalyzes a reaction Specificity change the way peptides bind to certain substrates No effect o Loss of enzymatic function Mechanisms of repair o Mutations that occur during DNA replication are repaired when possible by proofreading by DNA polymerases o Mutations that are not repaired by proofreading are repaired by mismatched post replication repair followed by excision repair o Mutations that occur spontaneously any time are repaired by excision repair base excision or nucleotide excision Repair of damaged DNA o Stress does not cause mutations usually something needs to happen for a mutation to occur o Thymine dimmer formation defect is usually lethal if not repaired o Photoreactivation two nucleotides on the same strand become base paired use light to break apart the thymine dimmer molecules can also use excision repair o SOS repair different DNA polymerase tries to come down and fix the mistake A test for carcinogens AMES test o His Salmonella strain of salmonella that is deficient in making its own histidine can t make its own amino acid histidine o Known mutation rate of that organism o Mix cells from the salmonella culture liver extract and chemical why liver extract A lot of things in our body do not become carcinogens until they hit our liver o Count his colonies compared to the control plate on media without histidine and that way strains that were not mutated do not survive mutations have to be in the genes that are responsible for histidine biosynthesis If you increased the mutation rate of the salmonella organism than it is a possible carcinogen o Mutagens carcinogens o Result the chemicals tested are carcinogens o Strain starts off histidine mutation is going to histidine Mutations happen only in genes Answer False o Why Can happen anywhere in the DNA sequence we just focus on gene mutations because if a mistake happens in a gene it affects the amino acid sequence which affects the protein o Can affect promoter sites operator sites origins of replication etc Horizontal gene transfer in bacteria o A major mechanism for spread of antibiotic resistance in bacteria Bacterial genetics horizontal gene transfer o How bacteria can acquire new DNA new genes new alleles of genes already present in bacterial genome o Transformation uptake of naked DNA into cell Linear DNA can do this too but unlike circular it cannot replicate unless put back into chromosome o Conjugation one cell has a pilus that connects it to another cell then DNA can become transferred into the new cell run it through a pilus o Transduction use of phage to put genes into a bacterium phage insert their DNA into cells Bacterial transformation free DNA o Transfer of genes from one bacterial cell to another by a mechanism involving o Competence ability to take up DNA and become genetically transformed o Natural competence rare some bacterial species have innate ability to take up DNA from the environment Bacillus streptococcu hemophillus pseudomonas neisseria o Induced competence ex e coli specially treat e coli in lab to make it competent Artificial or induced competence most bacterial species can be FORCED to take up DNA in the lab Electroporation use electrical charge to move DNA into cell charge disrupts the membrane the membrane then reforms with new DNA in it CaCl2 treatment used in lab divalent cat ions make membranes more permeable Gene guns shoot pellets covered with DNA into cell cell membrane reforms with new DNA in cell Griffith s experiment transformation o Strain R live cells took up DNA from the dead cells and gained the ability to make capsules See figure of experiment F transfer has conjugation pilus and can transfer the f plasmid which can encode a multitude of things such