Gene expression control inprokaryotes1. Single-celled organisms with short doubling times must respond extremely rapidly to their environment. 2. Half-life of most mRNAs is short (on the order of a few minutes).3. Coupled transcription and translation occur in a single cellular compartment. Therefore, transcriptional initiation is usually the major control point.The different consequences of activator and repressor binding to operator DNA.The metabolism of lactose in E. coli.To use lactose as an energy source, cells must contain theenzyme b-galactosidase.Utilization of lactose also requires the enzyme lactosepermease to transport lactose into the cell.Expression of these enzymes is rapidly induced ~1000-foldwhen cells are grown in lactose compared to glucose.Reactions catalyzed by b-galactosidaseNatural inducerArtificial inducer; can’t be cleavedHydrolysisTrans-glycosylationA simplified lac operon model.1. Polycistronic mRNA: The threegenes Z, Y, and A are coordinatelyexpressed.2. Negative regulation: The productof the I gene, the repressor, blocksthe expression of the Z, Y, and Agenes by interacting with theoperator (O).3. The inducer can inactivate therepressor, thereby preventinginteraction with the operator.Whenthis happens, the operon is fullyexpressed.How do we know that lacI encodes a trans-acting repressor?DNA binding by tetramers of lac repressorThe lac operator sequence is a nearlyperfect inverted repeat centered aroundthe GC base pair at position + 11.A simplified lac operon model.How can other inputs affect this system? Is it only a simple on/off switch?Catabolite control of the lac operon.(a) Under conditions of high glucose, a glucose breakdown product inhibits the enzyme adenylate cyclase,preventing the conversion of ATP into cAMP. (b) Under conditions of low glucose, there is no breakdown product, and therefore adenylate cyclase isactive and cAMP is formed.(c) When cAMP is present, it acts as an allosteric effector, complexing with CAP. (d) The cAMP–CAPcomplex acts as an activator of lac operon transcription by binding to a region within the lac promoter.(CAP = catabolite activator protein; cAMP = cyclic adenosine monophosphate.)X-ray structure of CAP-cAMP bound to DNACooperative binding of cAMP-CAP and RNAP on the lac promoterPositive and negative regulation of the lac operonThe trp operon: two kinds of negative regulationTryptophan + aporepressorTrp.repressor complex activated for DNA bindingBinds Operator; represses transcriptionAttenuation in the trp operonUUUPtrpEDCBA trpLTranscription pausesTranscription beginsAttenuation regulation of the trp operon of E. coli(Jackson, Bertrand, Lee, Squires, Squires, Platt, Korn, Zurawski, Landick, Stauffer, Winkler, Stroynowski, Das, Kolter, Fisher, Elseviers, etc)PRN A po lyme raseTranslation beginsTrpL-tRNAR N A p o ly m e ra s etrpEDCBA pThe moving ribosome releases the paused polymeraseTrpL-tRNAtrpEDCBA pAdequate Charged tRNATrpTranslating ribosomereaches the stop codonTranscriptionis terminatedTerminator formsTrpL-tRNAUGAtrpEDCBA p UGGTrpL-tRNACharged tRNATrp deficiencyAntiterminator formsRibosome stalls at one of two Trp codons Polymeraseresumes transcriptionMain Points1. Rapid mRNA turnover means that transcription likely to be a major step in gene expression control for prokaryotic organisms2. Lac gene control involves both positive and negative regulators: an inducible repressor binds to the operator and a cAMP dependent activator binds to the promoter3. Cooperative DNA binding by cAMP-CAP and RNA Pol for activation versus steric hindrance between lac repressor and RNA Pol for inhition4. Trp operon- an elegant mechanism that couples transcription and translation for feedback control5. Trp uses two distinct negative control mechanisms: Trp repressor and trp-dependent attenuation6. Linking ribosome movement and RNA secondary structure to transcription elongation and terminationcIORPRPLOLcI represses transcription fromdivergent operators and promotersProtein : Protein InteractionsBetween Dimers CooperativityHigher order interaction between repressors bound at distantoperators helps stabilize DNA-protein interactions further!MAIN POINTS ON THE LAMBDA REPRESSOR1. The lambda repressor is a dimer, with its DNA-bindingdomain in the N-terminal portion of the protein and itsdimerization domain in the C-terminal portion.2. The operator is a palindrome of two half operators to whichthe two subunits of the repressor bind.3. The lambda promoter PR is controlled from two adjacentoperators OR1 and OR2.4. Binding of repressor to OR1 and OR2 is facilitated byprotein-protein interaction.5. This “cooperativity” increases the specificity of
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