Class Note for ECOL 320 at UA
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Date Created: 02/06/15
Regulation of Gene Action The basis of cell differentiation is gene regulation different sets of genes are turned on and off in different cells There are other mechanisms as well but this is our focus Eg globin genes are expressed only in erythroblasts and are turned off in muscle cells Myosin genes are on in muscle cells but off in erythrocytes Progression through the cell cycle also requires turning different sets of genes on and off at different stages Bacteria and singlecelled eukaryotes undergo cell differentiation This includes responding to the availability of different nutrients I will discuss some of the most basic aspects Dr Restifo will give more detail Levels of Expression and Control The expression of any gene begins with transcription which can be regulated The expression of proteincoding genes requires several additional steps and can be turned off at any step However I will focus only on the control of transcription DNA transcription mRNA stability translation mRNA ribosomes tRNAs amino acids etc folding Polypeptide stability aggregation localization Protein Transcription Control in Prokaryotes Negative Regulation Negative regulation involves a protein repressor that binds to a repressor binding site and prevents binding of the transcription complex Inducible system off unless inducer molecule binds to and inactivates repressor Repressible system on unless co repressor binds to inactive aporepressor to form active repressor A Negative regulation of transcription Repressor binding site 1 Transcription B Inducible transcription Repressor 5 lg X No transcription L aim x n2 C Repressible transcription HI Aporepressor N Transcription Corepressor 1 Transcription gt41 Inactive repressor r i l I Active No transcription repressor Negative Regulation Examples Inducible system lactose operon in E 601139 E 601139 can cleave lactose into glucose galactose to use for carbon and energy sources This requires the enzyme 3 galactosidase and also galactoside permease to import the lactose into the cell If there is no lactose in the medium E 601139 does not make either S galactosidase or galactoside permease Synthesis is blocked at the transcription level If lactose is added to the medium the synthesis of both molecules is induced lac Operon Operon encodes lacZ B galactosidase Which cleaves lactose into glucose and galactose 0 lach lactose permease Which brings lactose into the cell 0 lacA thiogalactoside transacetylase not required for growth on lactose unknown function but sequence is conserved so it is important Other important players 0 lozch Promoter binds RNA polymerase to start transcription 0 lacO Operator binds repressor lac Repressor gene encodes repressor protein B galactosidase and lactose permease are made only if lactose is present only if they are neededThey are induced by their substrate A Promoter Repressor Operator gene Structural genes i z 1 E l gt Direction of transcription B i p o z y a ll i a 2 39 Repressor prevents mRNA transcription l a J Repressor quot protein c i p o z y a i a a lnducerrepressor complex K mRNA 39 i lnducer Cl J y K Repressor l39 protein Transcription allowed lac mRNA l l W Bgalactosidase Permease Transacetylase Simple Model of lac System A Promoter Repressor Operator gene Structural genes i z 39 Direction of transcription B p K o z y a 4 Repressor prevents mRNA transcription J Hg Repressor 1 protein C i p o z y a lnducer repressor complex mRNA 2 K 7 i i Inducer Repressorquot 39 39 protein IacmRNA Bgalactosidase Permease Transacetylase 16101 PO Z Y A Repressor protein 0 Inducer 0 r ii RNA polymerase lac Operon Mutants Basic features of luc operon were deduced from mutant phenotypes by Francois Jacob Jacques Monod and collaborators in 1960s by studying the phenotypes of mutants Jacob in medical battalion in North Africa wounded in action Commandant Malivert of the Resistance Legion of Honor Francois Jacob Jacques Monod Andre Lwoff Nobel Prize 1965 lac Operon Mutants Genotype Nature lacZ 39 Null mutation in lacZ lacY 39 Null mutation in lacY lacO can t bind repressor C taco constitutive always on Promoter can t bind RNA lacP 39 polymerase operon not transcribed lad S Repressor can t bind inducer super repressor Studying Interactions of lac Operon Mutants Studied the interactions of different mutant alleles in partial diploids which have the bacterial chromosome plus a plasmid with some genes Plasmids small DNA molecules that use their own replication origins to replicate independently of the cell chromosome have the own origin of replication Usually not required for cell function some may be present in many copies DNA molecules not 0 to scale F lac plasmid Chromosome lac Operon lac Operon lacZ lacY lacz lacy 39 Cell genotype F lacZ 39 lacY lacZ lacY 39 Cell phenotype Lac NOTE other genes assumed to be Wild type if not specified Phenotypes of lac Operon Mutants in Diploids Synthesis of Genotype lac mRNA Lac phenotype v F WUMT lacOlncZ Constitutive 2 F 1150 1152 lacotlacz39 Consmuuve 3 F MP162 lac39lacZJr lndudble 4 F hull IIMZ39 laLI IaCZ39 inducible 5 F lacOLlch lacO lch39 lndudble l 6 F 1150 ia Z IUCO IacZ Constitutive 7 F lucl lacz Inch162 Umndudble 8 F lacWacZ39 ladsac Unmducwble 9 F lacP39 lacZ IacP lucz nducible V0 F MCP39L aEZJr tchquotlz1rZ nducwble H F lacP lacZ IMP39 luLZ Unmducible 7 12 F lad a ZJr lacF IncZ39 lnducwble J acob Monod and collaborators deduced how the lac operon is controlled from these data and from the map position of the mutants Note 1010 and IacP mutants only affect expression of lac gnes on the same chromosome while lac mutants can operate at a distance from another chromosome lac Operon Second Level of Regulation Things are always more complicated than we d like When glucose is present 3 galactosidase etc are not made CAMP cyclic AMP CRP CAMP receptor protein Glucose inhibits synthesis of CAMP cAMPCRP must be bound to promoter in order for lac transcription i p o y a Flepressor y a CAMP CRP complex i p o y a gt Transcription I p o y a Synthesis of lac mRNA NO NO YES NO lac Operon Structural Details Beginning of 302 coding sequence Protected by RNA polymerase Ribnsume binding Protected by repressor site Met Thr Symmetrical operarar halves 1D II II GT TT AA GCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAA I II III II J b mRNA i 1 I360 Negative Regulation Examples Chorismic acid AS 2 E Indu01ble lac operon operon turned on I I ase lt r17 only When lactose is available as a Anthramllc 2101 l PRTase 4 trpD carbon and nitrogen source p A lnGPSase 4 tr C RepreSSIble system tryptophan C3121 p operon in E coli IHGP TSaseB 4 11773 Tryptophan is needed all the time by Ind ltTSaseA WA growing cells so lrp genes should be on all the time until cells have made sufficient tryptophan or it is provided in the medium L Tryptophan Negative Regulation Examples Repressible system tryptophan operon in E coli Tryptophan is needed all the time by growing cells so trp genes should be on all the time until cells have made sufficient tryptophan trpAE genes are in an operon OK if only needed to make tryptophan Has operator trp 0 and promoter trpp and attenuator trp a trp leader region Number of base pairs not drawn to scale in indicated region L 60 162560 1593 1350 1196 8011 300 A I v v v v DNA frp p frp o trpE trpD quotPC frpB trpA Spacer Attenuator Spacer sequence Irp a a Regulation T Enzyme production Negative Regulation of trp Operon Tryptophan levels low aporepresssor protein complex encoded by distant genes can t bind to promoter and transcription is on Tryptophan levels high tryptophan binds to aporepressor to form active repressor which binds to promoter and shuts off transcription Attenuation mechanism stops transcription in leader region unless there is sufficient TrptRNA in the cell provides finetuning of tryptophan synthesis A Transcription occurs trp p m o trpL trpE trpD trpC if 39 1 Transcription Aporepressor B Transcription is repressed trp 0 WP trpL trpE trpD trpC f Tryptophan r Active aporepressor No transcription
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