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1、Anticipatory Questions,1.What might happen if an organism had its cells expressing all genes within the genome all the time?2.At what levels can control of cellular activities/pathways be controlled?3.Based on our discussions up to this point,what do you think the term“negative feedback”means?4.What
2、 steps are involved in the initiation of prokaryotic transcription?,Learning Objectives,understand that regulation of gene expression is a means by which to control timing and rate of generation regarding functional gene product(either RNA or polypeptide/protein).explain the concept of an operon in
3、terms of components functions(promoter,operator,repressor,co-repressor,inducer,gene cluster,polycistronic transcript).compare and contrast repressible and inducible operon systems/pare and contrast negative versus positive regulation of operons apply the operon concept to gene expression as it relat
4、es to genetic engineering(specifically,our cloning and expression of the“tomato”gene).,Individual bacteria respond to environmental change by regulating their gene expression,A bacterium can tune its metabolism to the changing environment and food sourcesThis metabolic control occurs on two levels:A
5、djusting activity of metabolic enzymesRegulating genes that encode metabolic enzymes,LE 18-20,Regulation of enzymeactivity,Regulation of enzymeproduction,Enzyme 1,Regulation of gene expression,Enzyme 2,Enzyme 3,Enzyme 4,Enzyme 5,Gene 2,Gene 1,Gene 3,Gene 4,Gene 5,Tryptophan,Precursor,Feedbackinhibit
6、ion,Operons:The Basic Concept,In bacteria,genes are often clustered into operons,composed ofAn operator,an“on-off”switchA promoterGenes for metabolic enzymesAn operon can be switched off by a protein called a repressorA corepressor is a small molecule that cooperates with a repressor to switch an op
7、eron off,LE 18-21a,Promoter,Promoter,DNA,trpR,Regulatorygene,RNApolymerase,mRNA,3,5,Protein,Inactiverepressor,Tryptophan absent,repressor inactive,operon on,Polycistronic*mRNA,trpE,trpD,trpC,trpB,trpA,Operator,Start codon,Stop codon,trp operon,Genes of operon,E,Polypeptides that make upenzymes for t
8、ryptophan synthesis,D,C,B,A,5,*=mRNA carries the information of several genes,which are translated into several proteins,LE 18-21b_1,DNA,Protein,Tryptophan(corepressor),Tryptophan present,repressor active,operon off,mRNA,Activerepressor,LE 18-21b_2,DNA,Protein,Tryptophan(corepressor),Tryptophan pres
9、ent,repressor active,operon off,mRNA,Activerepressor,No RNA made,Trp Operon Animation,http:/,Repressible and Inducible Operons:Two Types of Negative Gene Regulation,A repressible operon is one that is usually on;binding of a repressor to the operator shuts off transcriptionThe trp operon is a repres
10、sible operonAn inducible operon is one that is usually off;a molecule called an inducer inactivates the repressor and turns on transcriptionThe classic example of an inducible operon is the lac operon,which contains genes coding for enzymes in hydrolysis and metabolism of lactose,LE 18-22a,DNA,lacl,
11、Regulatorygene,mRNA,5,3,RNApolymerase,Protein,Activerepressor,NoRNAmade,lacZ,Promoter,Operator,Lactose absent,repressor active,operon off,LE 18-22b,DNA,lacl,mRNA,5,3,lac operon,Lactose present,repressor inactive,operon on,lacZ,lacY,lacA,RNApolymerase,Polycistronic mRNA,Protein,Allolactose(inducer),I
12、nactiverepressor,-Galactosidase,Permease,Transacetylase,5,Lac Operon Animation,http:/,Inducible enzymes usually function in catabolic pathwaysRepressible enzymes usually function in anabolic pathwaysRegulation of the trp and lac operons involves negative control of genes because operons are switched
13、 off by the active form of the repressor,Positive Gene Regulation,Some operons are also subject to positive control through a stimulatory activator protein,such as catabolite activator protein(CAP)When glucose(a preferred food source of E.coli)is scarce,the lac operon is activated by the binding of
14、CAPWhen glucose levels increase,CAP detaches from the lac operon,turning it off,LE 18-23a,DNA,cAMP,lacl,CAP-binding site,Promoter,ActiveCAP,InactiveCAP,RNApolymerasecan bindand transcribe,Operator,lacZ,Inactive lacrepressor,Lactose present,glucose scarce(cAMP level high):abundant lacmRNA synthesized
15、,LE 18-23b,DNA,lacl,CAP-binding site,Promoter,RNApolymerase cant bind efficiently,Operator,lacZ,Inactive lacrepressor,InactiveCAP,Lactose present,glucose present(cAMP level low):little lacmRNA synthesized,Catabolite Activator Protein Mechanism,http:/highered.mcgraw-on“combination of switches-the lac
16、 operon”,The Arabinose Operon-A Composite of Negative&Positive Regulation,a)In the presence of arabinose:CAP-cAMP complex and araC-arabinose complex bind to initiator region this allows RNA polymerase to bind to the promoter transcription begins,b)In the absence of arabinose:araC protein assumes a d
17、ifferent conformationacts as a repressor binds to araI and a second operator region araO forms a loop this loop prevents transcription,Regulatory gene,Operator(part of the promoter),Promoter for the cluster of genesB,A,and D,Application of Operons:,araC regulatory gene,repressor,Inducer(arabinose),Gene D,Arabinose operon with in-frame foreign DNA inserted:,Tomato gene,start,start,start,start,stop,stop,stop,stop,Protein B,Protein A,Protein D,Red Fluorescent Protein(RFP),Gene B,Gene A,Gene D,Tomato gene,5,3,Polycistronic mRNA,transcription,translation,translation,translation,translation,
