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BIOLOGY 1A03 (168)
Xudong Zhu (12)
Chapter 17

Textbook and Class Notes Collaborated - Unit 3 - Chapter 17..
Textbook and Class Notes Collaborated - Unit 3 - Chapter 17 Bio 1A03

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McMaster University
Xudong Zhu

Bio 1A03 Unit Three: Gene Structure and Expression Chapter 17: Control of Gene Expression in Bacteria Key Concepts  Gene expression can be controlled at three levels: o Transcription o Translation o Post-Translation (protein activation)  Transcriptional control by a regulatory protein can be negative or positive o Negative control prevents transcription o Positive control increases the transcriptional rate  Many regulatory proteins bind to specific sites in DNA o Each regulatory protein has a distinct amino acid sequence so each binds to different DNA sequences 17.1 Gene Regulation and Information Flow  Gene expression – occurs when a gene product is actively being synthesized and used in a cell  Regulation of gene expression is critical to the efficient use of resources and so to survival  Escherichia coli has served as an excellent model organism for the study of prokaryotic gene regulation  Transcription and translation of genes in bacteria were predicted to be triggered by specific signals from the environment 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 o DNA  mRNA  protein  activated protein  Genes can be under transcriptional, translational or post-translational control  All three types of regulation occur in bacteria 1. Transcriptional Control – is slow but efficient  DNA  mRNA  protein  activated protein  The cell could avoid making the mRNAs for particular enzymes  If there is no mRNA, then ribosomes cannot make the gene product  Eg/ 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 transcriptional control  Saves the most energy for the cell, because it stops the process at the earliest possible point 2. Translational Control – allows a cell to quickly change which proteins are produced  DNA  mRNA  protein  activated protein  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 Ribonuclease, that affect translation initiation, or that affect elongation factors and other proteins during the translation process are forms of translational control 3. Post-Translational Control – provides the most rapid response but is energetically expensive  DNA  mRNA  protein  activated protein  Some proteins are manufactured in an inactive form and have to be activated by chemical modification, such as the addition of a phosphate group  The level of expression of different genes can be highly variable – some genes are: Bio 1A03 o Constitutively transcribed (are always expressed) o Regulated and their expression may be induced or repressed Metabolizing Lactose – A Model System  Glucose is the preferred carbon source for E. coli o Uses lactose only when glucose is depleted  E. Coli must transport lactose into the cell, where it can be cleaved by the enzyme β-galactosidase to produce glucose and galactose  E. Coli produces high levels of β-galactosidase only when lactose is present in the environment o β-galactosidase is not produced in E. coli cells grown in medium containing glucose or galactose  Lactose acts as an inducer of expression of β-galactosidase in E. coli cells o Inducer – a molecule that stimulates the expression of a specific gene or genes  Glucose acts as a repressor of expression of β-galactosidase in E. coli cells 17.2 Identifying the Genes Involved in Lactose Metabolism  Goal – to find genes that code for β-galactosidase and proteins that transport lactose into cells  Isolating mutants with respect to a particular trait is a two-step process  First step – generate a large number of individuals with mutations at random locations in their genomes  Second Step – use genetic screening on the mutants to find individuals with defects in the process or pathway in question  Genetic Screen – a technique that allows researchers to identify individuals with a particular type of mutation How Were the Genes Found?  Replica Plating and growth on indicator plates were key techniques in the search for mutants with defects in lactose metabolism  Replica Plating o Begins by spreading mutagenized bacteria on a plate that is filled with gelatinous agar containing glucose – master plate o Bacteria are allowed to grow – each cell produces a single colony, which consists of a large number of identical cells o A block covered with a piece of sterilized velvet is pressed onto the master plate, and cells from each colony are collected onto the velvet o The velvet is then pressed onto a plate containing a medium that differs from the master plate only by a single component o Cells from the velvet stick to the plates surface, producing an exact copy of the colonies on the master plate – replica plate Bio 1A03 o An investigator can compare the colonies that thrive on the replica plate medium with those on the master plate  First they grew bacterial colonies on master plates containing a medium with many sugars  They then replica plated these colonies to plates containing a medium with lactose as the only sugar to screen for colonies that could not grow on lactose  Colonies that grow on the master plate but are missing from the replica plate represent mutants deficient in lactose metabolism  They also used indicator plates, which allowed them to observe mutants with metabolic deficiencies directly o Added a compound that is acted on by β-galactosidase – compound acts as an indicator molecule for the presence of functioning β-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 β-galactosidase enzyme or its production o Made it possible for them to identify colonies without functioning β-galactosidase Different Classes of Lactose Metabolism Mutants  They isolated three classes of E. coli mutants defective in lactose metabolism Two Proteins are  They name the genes involved in lactose metabolism lacZ, lacY Critical for E. coli Cells to use Lactose and lacI  LacZ mutants lack functional β-galactosidase – the gene that encodes β-galactosidase is defective o Mutant unable to cleave the indicator molecule even if lactose was present inside the cells to induce production of the β-galactosidase protein  LacY mutants lack the membrane protein galactoside permease and so cannot transport into the cell o Cells failed to accumulate lactose inside the cell o Normal cells – the concentration of lactose is about 100 times that of lactose in the surrounding environment o Mutant cells – lactose concentrations were much lower  LacI mutants do not properly regulate production of β-galactosidase and galactoside permease, producing them even what lactose is absent o Did not regulate expression of β-galactosidase and galactoside permease normally Bio 1A03 o Grown on lactose alone – they turned yellow just as normal cells do when the indicator molecule was added o Grown on a medium that contained glucose but no lactose – still turned yellow when the indicator was added (normal cells remain white when grown on glucose) o Cells such as lacI mutants that are abnormal because they produce a product at all times are called constitutive mutants Several Genes Are Involved in Metabolizing Lactose  The lacZ and lacY genes code for proteins involved in lactose metabolism, while the lacI gene product serves a regulatory function  When lactose is absent – the lacI gene product shuts down expression of lacZ and lacY  When lactose is present – transcription of lacZ and lacY is induced  The physiFigure 17-5n of the three genes on E. coli’s circular chromosome are close together  The lacI gene controls both lacZ and lacY The lac GenesAre in Close Physical Proximity ! 17.3 Mechanisms of Negative Control: Discovery of the Repressor  Transcription can be regulated via negative control or positive control o Negative Control – occurs when a regulatory protein binds to DNA and shuts down transcription Figure 17-6blactosidase!Galactoside permease! Figure 17-6a o Positive Control (inductilacl product!when a regulatory prlaPositive regulation of gene expression! transcription Negative regulation of gene expression! Negative Regulation of Gene Expression lacl! Positive RegullacZ! of lacY!Expression No positive No transcription! No negative TRANSCRI
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