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BIOLOGY 1A03 (168)
Chapter 17

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McMaster University
Lovaye Kajiura

Chapter 17  Gene expression: is said to occur when a protein or other gene product is synthesized and is active in the cell  Why is understanding how bacterial cells help regulate gene expression funcdamental issue in biological science 1. Bacteria are the most abundant organisms on Earth ....Occupy virtually every habitat known 2. Questions about bacterial gene expression have enormous practical significance. ( biologists, are trying to understand which genes are expressed when disase- causing bacteria colonize a person and start an infection) - they can respond quickly to dramatic changes in temperature , pH, light, competitors, and nutrients - changes in gene expression give bacteria the ability to cope with these types of env changes Gene Regulation and Information Flow  Bacteria are found on every habitat on earth  Gene expression can be controlled at three levels; translation, transcpriton or post translation (protein activation) Mechanisms of Regulation---An overview  Gene expression can be controlled at any step between the synthesis of RNA and the activation of the final gene product  DNA- mRNA-proteins -activated protein  From DNA to RNA is transcription,( which is making of messenger RNA). RNA  protein is translation ( ribosome’s read information in mRNA /use that info to synthesize a protein), and protein activated protein is post translational modification ( folding , adding of carbohydrates or lipid group or perhaps phophorylation  What are the three possible mechanisms? 1. The cell would avoid making the mRNAs for particular enzymes. If there is no mRNA, then ribosomes cannot make the gene product. For example, various regulatory proteins affect the ability of RNA polymerase to bind to a promoter and initiate transcription. Genes that are controlled in this way are said to undergo transcptional control DNA- mRNA-proteins -activated protein 2. If the mRNA for an enzyme has been transcribed, the cell might have a way to prevent the mRNA from being translated into protein. Mechanisms that alter the length of time an mRNA survives before it is degraded by ribonucleases, that affect translation imitation or that affect elongation factors and other proteins during the translation process are forms of translational control. DNA- mRNA-proteins -activated protein 3. Some proteins have been manufactured in an inactive form, and have to be activated by chemical modification such as addition of phosphate group DNA- mRNA-proteins -activated protein  Which of these occur in bacteria? Answer: all  Transcptional control saves energy the most because it stops early in the process , slow  Translational control is advantageous because it allows a cell to make rapid changes in its range of proteins  Posttranslational control provides most rapid response of all 3 mechanism, fast but uses a lot of energy  Enzymes that code for genes such that they code for glycolysis---are transcribed all the time or constitutively Metabolizing Lactose ---A model system  Glucose is E.Coli preferred carbon source, however , meaning that is the source of energy and carbon atoms that the organism uses most efficiently  E.coli produces high levels of B-galactosidase only when lactose is present in the environment  Lactose acts as an inducer  Inducer: is a molecule that stimulates the expression of a specific gene or genes  B-galatosice is produced only when lactose is present and glucose is not present proven by EXPERIMENT]  Identifying the genes involved in Lactose Metabolism  Cells that can’t use lactose must lack either B-galactosidase or the lactose-transporter protein  Genetic screen: a technique that allows researchers to identify individuals with a particular type of mutation  What is a way to find mutatnts with respect to a particular trait 1. Generate a large number of individuals with mutations at random locations in their genomes 2. Screen the mutants to find individuals with defects in the process or biochemical pathway (defects in lactose metabolism )  How were genes found?  Techniques to finding mutants with defects are  replica dating and growth on indicator plates  Replica dating: begins with spreading mutagenized bacteria on a plate that is filled with gelatinous agar containing bacteria on a plate that is filled with gelatinous agar containing glucose (plate is known as master plate )  Bacteria grow so that each cell produces a single colony  Steps to Replica Dating 1. Grow a master plate of mutagenized E.Coli colonies on complete medium ( contains glucose as energy source ) 2. Press velvet covered block against master plate. Some cells from each colony transfer from plate to block. 3. Press block against a replica plate containing only lactose as an energy source. Location of cells on master plate and replica plate are an exact match. 4. After incubation, cells that can use lactose as energy source grow into colonies. Pick cells on master plate (which contain glucose only) that are not on replica plate. These cells are unable to metabolize lactose. *** The location of colonies on the master plate and replica plate must match exactly and the media in the two plates must differ by just one component. ****  Indicator molecule: where mutants with metabolic deficiencies are observed directly  He added a compound that is acted on by B-galactosidase  The compound acts as an indicator molecule for the presence of functioning B-galactosidase because one of the molecules produced by the reaction is yellow  Colonies that stay white are unable to process the indicator molecule, meaning they have a defect in the B-galactosidase enzyme or its production  Different Classes of Lactose Metabolism Mutants  LacZ cells cannot cleave (cut) indicator molecule even if lactose is present as an inducer o No B-galactosidase; gene for B-galactosidase is defective. Gene is called LacZ but genotype or allele is called LacZ-  LacYcells cannot accumulate (gather) lactose inside the cell o No membrane protein (galactosidase permease) required for import of lactose; gene for galactoside permease is defective. Gene is called LacZ but genotype or allele is called LacZ-  Lacl indicator molecule is cleaved even if lactose is absent ( no inducer) o Constitutive expression of lacZ and lacY; gene for regulatory protein that shuts down lacZ and lacY is defective (not working) ---it doesn’t need to be induced by lactose. Gene is called Lacl but genotype or allele is called lacl- ( didn’t express B-galactosidase and galactoside permease normally o EX) when they were grown on lactose alone and the indicator was added, it turned yellow just as normal cells do. But if they wer grown on a medium that contained glucose but no lactose, they turned yellow after the indicator molecule was added.  Constitutive mutants: cells that are abnormal because they produce a product at all times o I-didnt need an inducer to express B-galactosidase or galactoside permease o Mutants of lacI  gene expression occurred with or without lactose , so have a defect in gene regulation  Several Genes are Involved in Metabolizing Lactose  When lactose is absent the Lacl gene product, shuts down the expression of lacZ and lacY  When lactose is present , transcption of lacZ and lacy Y were induced Mechanisms of Negative Control: discovery of the Repressor  What are the two ways that transcption can be regulated?  Through negative control or positive control  Negative control: regulatotry protein shuts down transcption  Positive control: regulatory protein triggers transcption ,also called induction, occurs when a regulatoy protein binds to DNA  Repressor: the transcription inhibitor binds directly to DNA near or on the promoter for the lacZ and lacY genes  Lactose induces transcription by removing negative control  What is a repressor ? The repressor is the parking brake; lactose releases the parking brake , repressor codes for proteins that shut down transcption --- like parking brake (when repressor is present, B- galactosidase declines and transcption stops)  What happens when lactose removes the repressor? Lactose removes the repressor, it releases the parking brake  Negative control hypothesis: a) Repressor present, lactose absent  Repressor binds to DNA , no transcption occurs  Binds on or near the promoter for lacZ and lacY
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