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Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
Prokaryotic Gene Regulation
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Prokaryotic Gene Regulation

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  1. Prokaryotic Gene Regulation Beyond the Lac Operon
  2. Prokaryotic Genetics Tools <ul><li>The study of prokaryotic genetics exploits several biological processes: </li></ul><ul><ul><li>Bacterial Conjugation </li></ul></ul><ul><ul><li>Phage and Phage Transduction </li></ul></ul><ul><ul><li>Transformation </li></ul></ul><ul><ul><li>Cross-feeding </li></ul></ul><ul><ul><li>Complementation </li></ul></ul>
  3. Bacterial Conjugation <ul><li>Bacteria reproduce asexually </li></ul><ul><li>In order to increase diversity through genetic admixture, they have developed a mechanism to transfer of genetic material from one bacterium to another. </li></ul><ul><li>The ability to perform this transfer is conferred by a set of genes called “F” for fertility . </li></ul><ul><li>The fertility genes can exist on a plasmid (a small, circular piece of DNA that replicates independently from the bacterial chromosome), or they can be integrated into the chromosome. </li></ul>
  4. Transferring DNA <ul><li>Bacteria containing an “F” gene are called &quot;male&quot; </li></ul><ul><li>These bacteria shoot a grappling hook (called a pilus ) out to seize a neighboring bacterium. </li></ul><ul><li>The two cells are pulled together </li></ul><ul><li>The pilus forms a channel through which DNA is transferred. </li></ul><ul><li>This process is called bacterial conjugation </li></ul>
  5. Uses of Bacterial Conjugation <ul><li>Microbiologists can take advantage of bacterial conjugation to study prokaryotic genetics. </li></ul><ul><li>In Mendelian genetics, you analyze the function of a gene by looking at it as part of an allele pair </li></ul><ul><ul><li>Is the allele recessive or dominant? </li></ul></ul><ul><li>Prokaryotes are haploid organisms, so this analysis is impossible. </li></ul><ul><li>Pseudo-diploid ( meridiploid ) genetics can be conducted by putting a second allele of a gene of interest on the F element, with the fertility genes. </li></ul><ul><li>The gene of interest can then be transferred to another bacterium, which already contains a different allele of the gene. </li></ul><ul><li>The effect of the two alleles on each other can then be examined. </li></ul>
  6. Manifestations of the Fertility Factor Manifestation Description Hfr The F element is integrated into the genome. When conjugation occurs, the F genes travel across the pilus, dragging the rest of the genome behind it. Eventually, the pilus breaks, so most often the entire genome is not transferred. The bacterial genome can be measured in minutes from the origin of transfer: The amount of time it takes for a particular gene to be transferred from one bacterium to another indicates how far it is from the origin of replication. F' Also called the F' episome. This is a small circular piece of DNA that contains the fertility genes and a few other genes. These other genes are transferred very efficiently from one bacterium to the next because the length of the transferred DNA is short enough that it can move across the breach before the pilus breaks. F This is a small circular piece of DNA carrying only the fertility genes.
  7. Phage and Phage Transduction <ul><li>Microbiologists take advantage of the lysogenic cycle of phage to generate pseudo-diploid genetics in the haploid prokaryote. </li></ul><ul><li>Particular genes can be packaged into phage heads and these phage can be used to infect bacteria. </li></ul><ul><li>Phage strains have been constructed that cannot enter the lytic phase, so upon infection, the DNA integrates into the genome. </li></ul><ul><li>If the bacterium already has a copy of that gene, the two alleles' effect on each other can be examined. </li></ul>
  8. Transformation <ul><li>Bacteria can be induced to take up small pieces of circular DNA from the medium </li></ul><ul><li>Bacteria are treated with a solution of calcium chloride and then shocked at 42° C. </li></ul><ul><li>Why this protocol promotes absorption of DNA is not well understood, </li></ul><ul><li>A piece of DNA is constructed through recombinant DNA techniques to include genes of interest </li></ul><ul><li>This DNA can be transformed into bacterial cells containing another allele of the gene </li></ul><ul><li>The alleles' effect on each other can be examined. </li></ul>
  9. Cross-feeding <ul><li>When bacteria are grown on a petri dish they will secrete products they manufacture into the medium </li></ul><ul><li>If two bacterial strains that have different genotypes are plated near each other on the same petri dish, each will secrete substances into the medium. </li></ul><ul><li>If the product of the gene being studied can diffuse out of the cell, the product of the allele in one bacterial strain can affect the product of a different allele in the other strain. </li></ul>
  10. Pathways Involve Multiple Genes Both biosynthetic and catabolic pathways involve multiple enzymes and thus multiple genes
  11. Complementation <ul><li>To study prokaryotic genetics, scientists often mutate bacteria and look for mutants deficient in the pathway under study </li></ul><ul><li>How do you determine which mutants are affected in the same gene? </li></ul><ul><li>How do you determine how many different genes are involved in the pathway? </li></ul><ul><li>One method is a complementation test . </li></ul><ul><ul><li>If two bacteria are crossed to each other, and each is mutated in the same gene, function will not be restored. </li></ul></ul><ul><ul><li>If the mutations are in different genes, then the good copy of each gene in the other bacterium will fulfill the function of the mutated gene, or complement it. </li></ul></ul><ul><li>To cross haploid, asexually reproducing bacteria we use some of the techniques described on the previously to generate pseudo-diploid offspring </li></ul>
  12. A Complementation Example
  13. The Arginine Example <ul><li>There are several enzymes involved in arginine biosynthesis </li></ul><ul><li>We want to generate mutants in all of them. </li></ul><ul><li>Mutagenize bacteria and look for colonies that cannot grow unless arginine is supplied in the medium. </li></ul><ul><li>These bacteria are auxotrophic for arginine. </li></ul><ul><li>If we generate many mutants and cross them with each other, we can discover a series of complementation groups </li></ul><ul><li>That is, mutants whose mutations do not complement each other, and are thus in the same gene. </li></ul>
  14. Sample Results <ul><li>Mutants 1 and 4 can complement each other. </li></ul><ul><ul><li>Therefore they are in different genes. </li></ul></ul><ul><li>Mutants 1 and 2 do not complement each other. </li></ul><ul><ul><li>They are in the same gene and in the same complementation group. </li></ul></ul>
  15. Biochemical Pathways <ul><li>Now that we have a group of different mutants for arginine biosynthesis, can find the order of the genes in the pathway for arginine biosynthesis. </li></ul><ul><li>Given the following intermediates in the arginine biosynthesis pathway: </li></ul><ul><li>1 ---  2 ---  3 ---  Arginine </li></ul><ul><li>We now want to determine which of our three mutants corresponds to the enzyme that catalyzes each step in the pathway </li></ul>
  16. Identifying the Mutants <ul><li>We then place the mutants in media supplemented with various intermediates and test for growth: </li></ul><ul><li>The mutant that can use many intermediates to grow is mutated the earliest in the pathway. </li></ul><ul><li>The mutant that can use the least number of intermediates to grow is mutated latest in the pathway. </li></ul><ul><ul><li>The later in a pathway a mutant is blocked, the less can be done to rescue it. </li></ul></ul><ul><li>Thus we know that: </li></ul><ul><ul><ul><ul><ul><li> 1 ---  2 ---  3 ---  Arginine </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li> Arg1 Arg2 Arg3 </li></ul></ul></ul></ul></ul>
  17. Epistasis <ul><li>If we generated a bacterium that was deficient in both genes Arg1 and Arg3, the phenotype would be the same as Arg3 mutants </li></ul><ul><ul><li>The bacteria would not be able to grow on intermediate 2, because it is also lacking the enzyme Arg3. </li></ul></ul><ul><li>Thus, mutants in Arg3 are epistatic to mutants in Arg1 </li></ul><ul><ul><li>The phenotype of Arg3 masks that of Arg1. </li></ul></ul><ul><li>We can use this phenomenon to order elements of a pathway. </li></ul><ul><li>By generating double mutants, and discovering which phenotype is masked, we can order the genes in a pathway. </li></ul>
  18. Ordering Genes in a Pathway <ul><li>Our example: </li></ul><ul><li>The later a mutant is in a pathway, the more early mutants it can repress </li></ul><ul><li>That is, mask their phenotype by exhibiting its own. </li></ul><ul><li>Thus, we get the same result as before: </li></ul><ul><ul><ul><li>1 ---  2 ---  3 ---  Arginine </li></ul></ul></ul><ul><ul><li> Arg1 Arg2 Arg3 </li></ul></ul><ul><li>We can use the concept of epistasis to order genes in regulatory as well as biosynthetic pathways </li></ul>
  19. Why Gene Regulation <ul><li>If all cells have the same DNA content, and the DNA specifies the products made, why don’t all cells in an organism make the same proteins? </li></ul><ul><li>If DNA specifies cell characteristics (what effect enzymes have), why aren't all cells the same? </li></ul><ul><li>We know that there are different cell types in our bodies, and that the activities of cells change with time. </li></ul><ul><ul><li>The hormone-producing cells in the pituitary gland only produce growth hormone during childhood and adolescence. </li></ul></ul><ul><ul><li>These same cells remain in the pituitary in adulthood, but they don't function to produce growth hormone. </li></ul></ul><ul><ul><li>How do they know when they are needed or not needed? </li></ul></ul>
  20. Jacob and Monod <ul><li>First attempts to answer this were in simpler organisms </li></ul><ul><li>Francois Jacob and Jacques Monod examined single-celled prokaryotes like E. coli </li></ul><ul><ul><li>They worked on the lactose metabolism system in E. Coli . </li></ul></ul><ul><ul><li>They were the first scientists to elucidate a transcriptionally regulated system. </li></ul></ul><ul><li>Studied how E.coli are able to respond to changes in their environment </li></ul><ul><li>Each environmental cue generates a specific response, with specific proteins and reactions. </li></ul>
  21. Nitrogen Use Example <ul><li>A bacterium can use several different sources of nitrogen. </li></ul><ul><li>Some bacteria can incorporate diatomic nitrogen gas from the air, or incoprporate ammonia from their surroundings, or break the amine group from the end of an amino acid like glutamine. </li></ul><ul><li>It is easier and less costly for the cell to use the nitrogen from glutamine than to fix nitrogen gas from the air. </li></ul><ul><li>These two processes require different enzymes </li></ul><ul><li>If there is glutamine available, the cell should be able to shut off the enzymes used to incorporate nitrogen gas. </li></ul><ul><li>The organism shouldn't waste energy synthesizing these enzymes at all. </li></ul><ul><li>How can the cell &quot;turn off&quot; the synthesis of proteins from its DNA, when appropriate? </li></ul>
  22. Possibilities for Regulation <ul><li>The cell could selectively inhibit transcription of the gene. </li></ul><ul><ul><li>The mRNA for this gene would never be made. </li></ul></ul><ul><li>The cell could selectively degrade the mRNA as soon as it was made </li></ul><ul><ul><li>This would prevent it from being translated into protein. </li></ul></ul><ul><li>The cell could selectively prevent translation on an otherwise stable mRNA. </li></ul><ul><li>The cell could selectively degrade the translated protein </li></ul><ul><ul><li>The cell would not waste energy trying to catalyze reactions that it did not need </li></ul></ul>
  23. Transcriptional Regulation <ul><li>In terms of energy cost, it is better to shut off the process as early as possible </li></ul><ul><ul><li>Thus no energy is wasted in mRNA and protein synthesis. </li></ul></ul><ul><li>This type of early-intervention control is called transcriptional regulation </li></ul><ul><ul><li>Expression of the gene is regulated at the level of mRNA synthesis, or transcription. </li></ul></ul>
  24. Lactose Catabolism <ul><li>The system studied by Jacob and Monod </li></ul><ul><li>A transcriptionally regulated system. </li></ul><ul><li>When the bacterium is in an environment that contains lactose, the cell would want to turn on genes for enzymes that are required for lactose catabolism. </li></ul><ul><li>When lactose is absent, the cell would want to turn these genes off. </li></ul><ul><li>Actual regulation is more complicated than that. </li></ul><ul><li>The bacterium's prime source of food is glucose </li></ul><ul><ul><li>Glucose does not have to be modified to enter the repiratory pathway. </li></ul></ul><ul><ul><li>Catabolism of glucose is more energy favorable </li></ul></ul><ul><li>So if both glucose and lactose are available, the bacterium wants to turn off lactose metabolism in favor of glucose metabolism. </li></ul>
  25. The Lactose Enzymes <ul><li> -galactosidase : </li></ul><ul><ul><li>Hydrolyzes the bond between the two </li></ul></ul><ul><ul><li>sugars, glucose and galactose. </li></ul></ul><ul><ul><li>Coded for by the gene LacZ. </li></ul></ul><ul><li>Lactose Permease : </li></ul><ul><ul><li>A membrane-spanning protein </li></ul></ul><ul><ul><li>Brings lactose into the cell from the outside environment. </li></ul></ul><ul><ul><li>The membrane is otherwise impermeable to lactose. </li></ul></ul><ul><ul><li>Coded for by the gene LacY. </li></ul></ul><ul><li>Thiogalactoside transacetylase : </li></ul><ul><ul><li>The function of this enzyme is not known. </li></ul></ul><ul><ul><li>Coded for by the gene LacA </li></ul></ul>
  26. The Lac Operon <ul><li>The genes for these three enzymes appear adjacent to each other on the E. Coli genome. </li></ul><ul><li>They are preceded by a region which is responsible for the regulation of the lactose metabolic genes. </li></ul><ul><li>There are sites upstream of the lactose genes that respond to glucose concentration. </li></ul><ul><li>This assortment of genes and their regulatory regions is called the Lac operon . </li></ul>
  27. A Diagram of the Lac Operon
  28. Elements of the Lac Operon ELEMENT PURPOSE Operator (Lac O) Binding site for repressor Promoter (Lac P) Binding site for RNA polymerase Repressor (Lac I) Gene encoding lac repressor protein Binds to DNA at operator and blocks binding of RNA polymerase at promoter Pi Promoter for LacI CAP Binding site for cAMP/CAP complex
  29. And Another Diagram
  30. Lac Operon Action <ul><li>When lactose is present, it acts as an inducer of the operon. </li></ul><ul><li>Lactose enters the cell and binds to the Lac repressor </li></ul><ul><li>This induces a conformational change that allows the repressor to fall off the DNA. </li></ul><ul><ul><li>Now RNA polymerase is free to move along the DNA </li></ul></ul><ul><ul><li>RNA can be made from the three genes. </li></ul></ul><ul><ul><li>Lactose can be metabolized </li></ul></ul><ul><li>When the inducer (lactose) is removed, the repressor returns to its original conformation and binds to the DNA </li></ul><ul><ul><li>RNA polymerase can no longer get past the promoter. </li></ul></ul><ul><ul><li>No RNA and no protein is made. </li></ul></ul>
  31. Induction of the Lac Operon
  32.  
  33. <ul><li>RNA polymerase can still bind to the promoter, but it cannot move past it. </li></ul><ul><ul><li>Thus when the cell is ready to use the operon, RNA polymerase is already waiting to begin transcription </li></ul></ul><ul><ul><li>The operon is primed for transcription when lactose is added. </li></ul></ul>
  34. Catabolite Repression <ul><li>When levels of glucose (a catabolite ) in the cell are high, cyclic AMP is inhibited from forming. </li></ul><ul><li>When glucose levels drop, more cAMP forms. </li></ul><ul><li>cAMP binds to a protein called CAP (catabolite activator protein), which is then activated to bind to the CAP binding site. </li></ul><ul><li>This activates transcription, perhaps by increasing the affinity of the site for RNA polymerase. </li></ul><ul><li>This phenomenon is called catabolite repression , a misnomer since it involves activation </li></ul><ul><ul><li>The presence of glucose represses all the other sugar metabolism operons. </li></ul></ul>
  35. Action of CAP
  36.  
  37. Methods for Studying Lac Regulation <ul><li>Some important tools: </li></ul><ul><li>IPTG (isopropyl-beta-D-thiogalactoside) </li></ul><ul><ul><li>A molecule that looks like lactose to the Lac repressor (Lac I). </li></ul></ul><ul><ul><li>This molecule can be used as a gratuitous inducer </li></ul></ul><ul><ul><li>It will induce the Lac operon by altering the conformation of Lac I so that it no longer blocks the promoter, but it is not a substrate for the lactose metabolism genes. </li></ul></ul><ul><li>ONPG </li></ul><ul><ul><li>We can measure the amount of mRNA made on the Lac operon by measuring the amount of B-galactosidase activity. </li></ul></ul><ul><ul><li>B-galactosidase can be fooled into cleaving a colorless substrate called ONPG into a yellow product called ONP. </li></ul></ul><ul><ul><li>Thus we can use a replacement substrate with an obvious visible product to measure amounts of the enzyme. </li></ul></ul>
  38. Types of Lac Mutations <ul><li>Consider what mutations in various elements of the Lac operon could exist </li></ul><ul><li>A constitutively expressed gene (denoted c) is never turned off. </li></ul><ul><li>It is making mRNA and protein all the time. </li></ul><ul><li>An uninducible gene is never turned on. </li></ul><ul><li>An uninducible DNA binding site is mutated so that it never binds its protein. </li></ul><ul><li>A super-repressor can be denoted s . </li></ul><ul><li>This repressor always represses, regardless of its regulation. </li></ul><ul><li>For example, a Lac I(s) mutant always represses at the promoter regardless of whether or not lactose is present. </li></ul>
  39. Lac Operon Mutants GENOTYPE PHENOTYPE +IPTG -IPTG Wild Type + - Lac Z- - - Lac P- - - Lac O- + + Lac I- + + Lac I(s) - - Lac O-Lac I(s) + +
  40. Binding Sites vs. Proteins <ul><li>There are two different kinds of elements are present in the Lac operon's regulation: </li></ul><ul><ul><li>DNA binding sites: Lac P, Lac O, Pi </li></ul></ul><ul><ul><li>Proteins: CAP, Lac I, and Lac Z, Lac Y, and Lac A </li></ul></ul><ul><li>It is useful to differentiate between the two types of elements. </li></ul><ul><li>Examine the phenotypes of Lac O and Lac I mutants. </li></ul><ul><ul><li>They are the same. </li></ul></ul><ul><li>How would we be able to distinguish which one was a DNA-binding site and which one was a mutant protein? </li></ul>
  41. Cis and Trans <ul><li>We can use pseudo-diploid genetics to see if a DNA sequence can act from afar on another DNA sequence. </li></ul><ul><li>If it can, then it is a diffusible protein. </li></ul><ul><ul><li>These sites are called trans-acting sites, since they act from afar. </li></ul></ul><ul><li>If the site cannot act from afar, then it is a DNA binding site that needs to be near other DNA sites (such as coding sequences) in order to function. </li></ul><ul><ul><li>These sites are called cis-acting sites, since they need to be next to other DNA to work. </li></ul></ul>
  42. Diffusible Proteins Act Trans
  43. DNA Binding Sites Act Cis
  44. Does a Gene Acts Cis or Trans? <ul><li>In order to see if a DNA element is acting in cis or in trans to another DNA element, we can insert a piece of DNA carrying element 1 into a cell that already has a copy of mutated element 1 next to element 2. </li></ul><ul><li>If the inserted element can complement or replace the function of the mutated element, it is acting in trans </li></ul><ul><ul><li>It must diffuse off a plasmid or from another site in the DNA in order to be functional. </li></ul></ul><ul><ul><li>It must be a diffusible protein. </li></ul></ul><ul><li>If the two functional pieces of DNA must be adjacent to each other to work they are acting in cis . </li></ul><ul><ul><li>One must be a DNA binding site affecting the other. </li></ul></ul>
  45.  
  46. Results of the Cis-Trans Test <ul><li>Any pair of DNA elements that passes both the cis and trans test must be acting in trans </li></ul><ul><ul><li>It is therefore a coding region for a protein </li></ul></ul><ul><li>Any pair that passes only the cis test must be acting in cis </li></ul><ul><ul><li>It is therefore a DNA binding site </li></ul></ul><ul><li>There are some DNA binding elements that can act in trans, through folding and looping of the DNA strand, but these are largely in eukaryotes. </li></ul>
  47. Strategies for Understanding Regulation <ul><li>Find mutations that render the regulation uninducible or constitutive </li></ul><ul><li>Perform a complementation test to determine if the mutants are dominant or recessive </li></ul><ul><li>If they are recessive, decide if the system is regulated by repression or by activation. </li></ul><ul><ul><li>A recessive mutated activator has most likely lost function: the system will become uninducible. </li></ul></ul><ul><ul><li>A recessive mutated repressor has also lost function, but now the system will have constitutive expression. </li></ul></ul><ul><li>Decide if the elements of the system act in cis or in trans to each other </li></ul><ul><ul><li>Are they diffusible proteins or DNA binding sites? </li></ul></ul><ul><li>Construct a model </li></ul>
  48. Life After the Lac Operon <ul><li>The Lac Operon was the first well described example of prokaryotic gene regulation and one of the best understood </li></ul><ul><li>There are many other transcriptionally regulated systems in prokaryotes. </li></ul><ul><li>Some examples that demonstrate differing regulatory strategies include: </li></ul><ul><ul><li>The Tryptophan Operon: A Repressor </li></ul></ul><ul><ul><li>The Histidine Operon: An Attenuator </li></ul></ul><ul><ul><li>The Lambda Phage Cycle: Decision Control </li></ul></ul><ul><ul><li>Quorum Sensing: An Activator </li></ul></ul><ul><li>In considering regulation of a system we must first ask: </li></ul><ul><ul><li>Why is the bacterium regulating this system? </li></ul></ul><ul><ul><li>When should these genes be turned on and when should they be turned off? </li></ul></ul>
  49. The Tryptophan Operon: A Repressor <ul><li>When should bacteria transcribe genes for the synthesis of the amino acid tryptophan? </li></ul><ul><li>When levels of tryptophan in the cell are low, the bacteria must make its own. </li></ul><ul><li>If tryptophan is abundant in the cell or is provided in the medium, it is a waste of energy for the bacteria to synthesize it. </li></ul><ul><li>The Trp repressor protein can bind to the operator of the Trp operon, which contains the tryptophan biosynthetic genes. </li></ul>
  50. The Tryptophan Operon
  51. Regulating Tryptophan Synthesis <ul><li>When tryptophan is available, it binds to the Trp repressor </li></ul><ul><ul><li>This induces a shape change, so that the repressor can bind to the DNA. </li></ul></ul><ul><ul><li>The operator is blocked </li></ul></ul><ul><li>When tryptophan levels are low, the tryptophan falls off of the repressor </li></ul><ul><ul><li>The repressor goes back to its original conformation, losing its ability to bind to the DNA. </li></ul></ul><ul><ul><li>The operator is now free for RNA polymerase </li></ul></ul><ul><ul><li>Transcription proceeds, copying the tryptophan biosynthetic genes and replenishing the cell's supply of tryptophan. </li></ul></ul><ul><li>This type of feedback inhibition of transcription is very common. </li></ul>
  52. Repression of the Tryptophan Operon
  53.  
  54. The Histidine Operon: An Attenuator <ul><li>The histidine operon functions in a different way. </li></ul><ul><li>At the beginning of the operon there is a leader coding region with the following code and corresponding amino acid sequence: </li></ul><ul><ul><li>AUG-AAA-CGC-GUU-CAA-UUU-AAA-CAC-CAC-CAU-CAU-CAC-CAU-CAU-CCU-GAC </li></ul></ul><ul><ul><li>Met-Thr-Arg-Val-Gln-Phe-Lys- His-His-His-His-His-His-His -Pro-Asp- </li></ul></ul><ul><li>When this sequence begins transcription, the RNA comes off of the DNA and ribosomes hop onto it to start translation. </li></ul><ul><li>If there is little histidine in the cell, the ribosome stalls because there are no aminoacyl tRNA's that are charged with histidine. </li></ul>
  55. Attenuation and Termination <ul><li>The stalling ribosome leaves a long stretch of RNA (RNA polymerase is still transcribing it) with no ribosome bound </li></ul><ul><li>The sequence of this RNA allows it to form a terminator loop only when ribosomes are bound </li></ul><ul><ul><li>The RNA is then cleaved and the RNA polymerase stops transcribing the genes. </li></ul></ul><ul><li>So, the terminator only functions when the ribosome is not stalled </li></ul><ul><ul><li>That is, when there is already plenty of histidine in the cell. </li></ul></ul><ul><li>The site at which the potential terminator loop forms is called the attenuation site . </li></ul><ul><li>Many amino acid synthetic operons are controlled by some form of attenuation. </li></ul><ul><ul><li>The tryptophan operon has attenuation control as well as the repressor control described above. </li></ul></ul>
  56.                                                                         
  57. The Lambda Phage Cycle: Decision Control <ul><li>A bacteriophage can switch between lytic and lysogenic cycles </li></ul><ul><ul><li>When there are many bacteria available to infect, and conditions for growth are favorable, the phage will take advantage and replicate itself as much as possible. </li></ul></ul><ul><ul><li>When there are few bacteria around and little growth potential, the phage is better off integrating into the bacterial genome and waiting until conditions improve </li></ul></ul><ul><li>How does the phage make these decisions? </li></ul><ul><li>There are two competing proteins in the lambda bacteriophage. </li></ul><ul><ul><li>One protein, CI, promotes the lysogenic cycle. </li></ul></ul><ul><ul><li>The other protein, Cro, promotes the lytic phase. </li></ul></ul><ul><li>These two proteins are in direct competition to each other for sites on the &quot;right&quot; promoter of lambda: </li></ul>
  58.  
  59. And Glowing Squid? <ul><li>A particular species of squid swims at the top of the ocean at night, skimming for food. </li></ul><ul><li>Unfortunately, to any predator below, this squid appears as a very dark object moving against the very bright background of the moon. </li></ul><ul><li>To solve this problem, the squid has evolved a light organ </li></ul><ul><li>Inside the light organ is a dense culture of a bacteria called Vibrio fischeri. </li></ul><ul><li>This bacteria produces the protein luciferase, which glows with the same intensity as the moon, rendering the squid invisible to predators from the depths of the ocean. </li></ul>
  60. Regulating Luciferase Production <ul><li>When Vibrio fischeri is not in the squid's light organ, it does not need to make luciferase </li></ul><ul><ul><li>Glowing will not help either the bacteria or the squid survive. </li></ul></ul><ul><li>When inside the light organ, it is in the bacteria's best interest to glow </li></ul><ul><ul><li>The squid will not get eaten and will feed it, away from competition from other kinds of bacteria. </li></ul></ul><ul><li>So how can the bacteria know that it is in a light organ in order to turn on synthesis of luciferase? </li></ul>
  61. Quorum Sensing: An Activator <ul><li>When the density of bacteria is very high, the bacteria know that they must be inside a light organ instead of floating around in the ocean. </li></ul><ul><li>Each bacterium is continuously secreting a unique small molecule called VAI ( Vibrio fischeri autoinducer) that can diffuse readily through the cell membrane. </li></ul><ul><li>Thus, there is a declining concentration of the small molecule in a growing circumference around the bacterium. </li></ul><ul><li>When there are many bacteria around, the local concentration of the small molecule will be very high. </li></ul>
  62. The Lux Operon <ul><li>The genes for luciferase are contained in the lux operon. </li></ul><ul><li>A DNA binding site (lux O) near the lux promoter (lux P) binds a protein called lux R. </li></ul><ul><ul><li>This protein calls RNA polymerase over when it is bound to the DNA, increasing transcription of the DNA and making more polymerase. </li></ul></ul><ul><li>Thus, lux R is a transcriptional activator of the lux operon. </li></ul><ul><li>When the local concentration of VAI is very high, it binds to luxR, enabling it to bind the operator and turn on transcription. </li></ul><ul><ul><li>If VAI is low, the luxR shape cannot bind to the operator </li></ul></ul><ul><ul><li>Not much luciferase can be made. </li></ul></ul><ul><li>LuxR is consistently transcribed at a low level so that there is always some around to affect regulation. </li></ul><ul><li>In this fashion, the bacteria only make luciferase when there are lots of other bacteria around. </li></ul>
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  64. Production Without Inducer? <ul><li>LuxI is the gene that encodes for the enzyme that synthesizes VAI. </li></ul><ul><li>When a bacterium undergoes the transition from not making luciferase to making luciferase, it needs to have the autoinducer around in order to promote binding of LuxR to the operator. </li></ul><ul><li>But before the operon is turned on, how can LuxI be made so that there is a continous level of autoinducer being made? </li></ul><ul><li>Operons, in general, are never completely turned off. </li></ul><ul><ul><li>There is always some basal level of transcription going on, </li></ul></ul><ul><li>In this case, the uninduced LuxR protein still has a minimal affinity for the DNA binding site so that some DNA can be transcribed to make enough LuxI so that autoinducer is continuously made. </li></ul>
  65. Other Uses of Quorum Sensing <ul><li>We tend to think of bacteria as single celled organisms living completely independently from each other. </li></ul><ul><li>This isn't true. bacteria often communicate with other bacteria in the community. </li></ul><ul><li>For example, what would be the point of making proteins to carry out bacterial conjugation if there were no other bacteria around? </li></ul><ul><li>Quorum sensing is used to determine whether or not there are enough bacteria around to make it worth it to turn on the machinery for conjugation. </li></ul>

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