Overview of Gene Regulation. Prokaryotic gene regulation Key Concepts: Chapter 13 Gene Regulation

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1 Chapter 13 Gene Regulation verview of Gene Regulation Key Concepts: n verview of Gene Regulation n Regulation of Transcription in Bteria n Regulation of Transcription in Eukaryotes: Roles of Transcription Ftors and Mediator n Regulation of Transcription in Eukaryotes: Changes in Chroatin Structure and DNA Methylation n Regulation of RNA Processing and Translation in Eukaryotes 1 n Gene regulation refers to the ability of cells to control their level of gene expression n Majority of genes regulated to ensure that proteins are produced at the correct tie and aount n Saves energy by producing only when needed n Constitutive genes are unregulated and have essentially constant levels of expression 2 Prokaryotic gene regulation n Responds to changes in the environent Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. β-galtosidase: Breaks down E. coli DNA ltose. 1 Ltose becoes available in the environent of the bteriu. Ltose perease: Transports ltose into the cell. n ex: Escherichia coli and ltose q When ltose is available, two proteins are ade: n ltose perease transports ltose into the cell 4 Most proteins involved with the use of ltose are degraded. Ltose 2 Due to gene regulation, the bteriu increases production of the ltose perease and β-galtosidase proteins. n β-galtosidase breaks down ltose q When ltose levels drop, the proteins are no longer ade 3 The bteriu readily uses the ltose until it is gone

2 Gene regulation in eukaryotes Gene regulation occurs at different points Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. n Necessary to produce different cell types in an organis cell differentiation n All of the organis s cells contain the sae genoe but express different proteoes due to gene regulation q Different proteins q Different aounts of the sae protein n Bterial gene regulation q Most coonly occurs at the level of transcription q Also can control rate of translation q Can be regulated at protein or post-translation level Transcription Translation Gene Protein Functional protein Regulation (a) Bterial gene regulation 6 n Eukaryotic gene regulation q Transcriptional regulation coon q RNA processing q Translation q Post-translation Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. Transcription RNA processing Translation Posttranslation Posttranslation Gene pre- Protein Functional protein (b) Eukaryotic gene regulation Regulation 7 Regulation of Transcription in Bteria n Involves regulatory transcription ftors n Bind to DNA in the vicinity of a prooter and affect transcription of one or ore nearby genes n Repressors inhibit transcription q Negative control n Activators increase the rate of transcription q Positive control 8 2

3 Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. DNA Prooter Structural gene No transcription Repressor Negative control: A repressor inhibits transcription. RNA polyerase Activator Positive control: An tivator prootes transcription. Transcription occurs n Transcriptional regulation also involves sall effector olecules n Binds to regulatory transcription ftor and causes conforational change n Deterines whether or not regulatory transcription ftor can bind to DNA n Two doains in regulatory transcription ftor that respond to sall effector olecules q Site where protein binds to DNA q Site specifically for sall effector olecule (a) Actions of regulatory transcription ftors 9 peron Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. DNA A sall effector olecule RNA Prooter becoes present in the cell polyerase Structural gene No transcription Doain that Repressor Transcription occurs binds to DNA Doain that recognizes sall effector olecule Repressor Sall effector olecule The repressor protein is bound to the The binding of the sall effector olecule causes a prooter region when the sall effector conforational change in the repressor protein that olecule is not present. prevents it fro binding to the DNA. (b) Action of a sall effector olecule on a repressor n peron in bteria is a cluster of genes under transcriptional control of one prooter q Regulatory region called operator n Transcribed into as polycistronic encodes ore than one protein n Allows coordinated regulation of a group of genes with a coon function

4 l operon n In E. coli contains genes for ltose etabolis n lp l prooter n Three structural genes q lz β-galtosidase q ly ltose perease q la galtosidase transetylase n Near the l prooter are two regulatory sites q l operator provides binding site for repressor protein q CAP site tivator protein binding site n li gene codes for l repressor q Considered a regulatory gene since its sole function is to regulate other gene s expression q as its own prooter (not part of l operon) E.coli chroosoal DNA l regulatory gene li Encodes l i prooter repressor CAP site perator (l) l prooter (lp) Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. lz l operon Encodes β-galtosidase Transcription ly Encodes ltose perease la Encodes galtoside l transetylase terinator n When ltose is absent q L repressor protein binds to nucleotides of the l operator site preventing RNA polyerase fro transcribing lz, ly and la q RNA polyerase can bind but not ove forward Polycistronic (a) rganization of DNA sequences in the l region of the E. coli chroosoe E. coli + Ltose perease Ltose + β-galtosidase Ltose C 2 (b) Functions of ltose perease and -galtosidase C 2 Galtose + C 2 C 2 Glucose C 2 C 2 Alloltose 1 Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. When ltose is absent fro the environent, alloltose is not ade and the l repressor is free to bind to the l l operator. The transcription of the operon is inhibited. regulatory l operon gene RNA polyerase l prooter perator li lp l lz ly la L repressor binds to the operator and inhibits transcription. L repressor (tive) (a) Ltose absent fro the environent 4

5 n When ltose is present q Alloltose is a sall effector olecule 4 olecules bind to l repressor to prevent it fro binding DNA q Process is induction the l operon is inducible Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. L operon also under positive control by tivator protein n CAP (catabolite tivator protein) is an tivator li When ltose is present, alloltose is ade inside the cell. The binding of alloltose to the l repressor prevents it fro binding to the l operator site. This perits the transcription of the l operon. RNA polyerase n Catabolite repression glucose, a catabolite, represses l operon lp l lz ly la Transcription Polycistronic Translation n Sall effector olecule, camp, binds to tivator protein called catabolite tivator protein (CAP) or camp receptor protein (CRP) Alloltose β-galtosidase Ltose perease Galtoside transetylase n peron is turned off when CAP is not bound Conforational change (b) Ltose present The binding of alloltose to the l repressor causes a conforational change that prevents the l repressor fro binding to the operator site. L repressor (intive) 17 n Glucose inhibits production of camp and so prevents binding of CAP to DNA 18 n Gene regulation involving CAP and camp an exaple of positive control n When camp binds to CAP, coplex binds to CAP site near l prooter n Resulting bend in DNA enhances RNA polyerase binding which increases transcription Threediensional structure of CAP bound to the CAP site Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. CAP site CAP dier CAP site Binding of RNA polyerase to prooter is enhanced by CAP binding. camp DNA Prooter perator CAP camp Transcription occurs RNA polyerase Thoas Steitz, oward ughes Medical Institution, Yale University Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. CAP site Prooter perator Alloltose CAP (Intive) (a) Ltose high, glucose high L repressor (intive) Alloltose high, camp low Low rate of transcription n When both ltose and glucose are high, the l operon is shut off q Glucose uptake causes camp levels to drop q CAP does not tivate transcription Transcription is low due to a lk of CAP binding. q Bteriu uses one sugar at a tie, glucose 19 20

6 Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display.z CAP site Alloltose high, camp high Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. igh rate of Prooter perator transcription CAP camp Alloltose L repressor (intive) Binding of RNA polyerase to prooter is enhanced by CAP binding. Transcription rate is high. CAP site Prooter perator CAP site Prooter perator Alloltose low, Alloltose low, camp low camp high Very low rate of Very low rate of transcription transcription CAP camp Transcription is inhibited by lk of RNA polyerase binds, but CAP CAP binding and by the binding of transcription is blocked by the l repressor. the binding of the l repressor. (Intive) (c) Ltose low, glucose high (d) Ltose low, glucose low (b) Ltose high, glucose low n When ltose is high and glucose is low, the l operon is turned on q Alloltose levels rise and prevent l repressor fro binding to operator q CAP is bound to the CAP site q Bteriu uses ltose 21 n When ltose is low and glucose is high or low, the l operon is shut off q Under low ltose conditions, l repressor prevents transcription of l operon 22 Regulation of Transcription in Eukaryotes: Roles of Transcription Ftors and Mediator Cobinatorial control 1. Activators tivator proteins stiulate RNA polyerase to initiate transcription n Soe of sae principles as in prokaryotes q Activator and repressor proteins influence ability of RNA polyerase to initiate transcription q Many regulated by sall effector olecules n Many iportant differences q Genes alost always organized individually q Regulation ore intricate Repressors repressor proteins inhibit RNA polyerase fro initiating transcription 3. Modulation sall effector olecules, protein protein intertions, and covalent odifications can odulate tivators and repressors 4. Chroatin tivator proteins proote loosening up of the region in the chroosoe where a gene is located, aking it easier for RNA polyerase to transcribe the gene. DNA Methylation usually inhibits transcription, either by blocking an tivator protein or by recruiting proteins that ake DNA ore copt 24 6

7 Three features of ost prooters qtata box n TATAAAA 3 n 2 base pairs upstrea fro transcriptional start site n Deterines precise starting point for transcription qtranscriptional start site n Where transcription begins n With TATA box fors core prooter q By itself results in low level basal transcription qregulatory or response eleents n Recognized by regulatory proteins that control initiation of transcription n Enhancers and silencers Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display Coding-strand sequences: Coon location for regulatory eleents Prooter TATA box Core prooter Transcriptional start site TATAAA Py 2 C A Py +1 Structural gene Transcription Three proteins ediate transcription 1. An RNA polyerase II 2. different general transcription ftors (GTFs) 3. Large protein coplex called ediator Transcriptional start site TATA box Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. GTFs Preinitiation coplex n GTFs and RNA polyerase II ust coe together at core prooter before transcription can be initiated n Preinitiation coplex assebled GTFs and RNA polyerase II at the TATA box q For basal transcription apparatus n Mediator coposed of several proteins q Partially wraps around GTFs and RNA polyerase II q Mediates intertions with tivators or repressor that bind to enhancers or silencers q Controls rate at which RNA polyerase can begin transcription RNA polyerase II

8 Transcriptional regulation n Activators bind to DNA regions called enhancers n Repressors bind to DNA regions called silencers n Regulate rate of transcription of a nearby gene n Most do not bind directly to RNA polyerase II 3 ways to control RNA polyerase II 1. Activators and repressors regulate RNA polyerase II by binding to GTFs (including TFIID) 2. Regulate RNA polyerase II via ediator q Activators stiulate ediator by allowing faster initiation q Repressors inhibit ediator so RNA polyerase II cannot progress to elongation 3. Recruit proteins that influence DNA pking Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. An tivator binds to an enhancer. Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. Enhancer Activator Mediator Preinitiation coplex Enhancer 2 The tivator enhances the ability of a GTF called TFIID to bind to the TATA box. TFIID TATA box 3 TFIID prootes the assebly of the preinitiation coplex. TFIID Cotivator Preinitiation coplex TATA box 31 Enhancer Activator 32 8

9 Regulation of Transcription in Eukaryotes: Changes in Chroatin Structure and DNA Methylation n DNA is associated with proteins to for copt chroatin n Chroatin pking affects gene expression n Transcription is difficult or ipossible in the closed conforation of tightly pked chroatin n Access to the DNA is allowed in the loosely pked open conforation n Soe tivators diinish DNA coption near a gene n Recruit proteins to loosen DNA coption q istone etyltransferase atthes etyl groups to histone proteins so they don t bind DNA as tightly q ATP-dependent chroatin reodeling enzyes also loosen DNA coption Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. or Chroatin-reodeling coplex Changes the sping (not shown) changes the relative or of nucleosoes over a positions of a few nucleosoes. long distance. (a) Change in nucleosoe position istone octaers are reoved by a chroatin-reodeling coplex. (b) istone eviction Standard histones are repled with variant histones by a chroatin-reodeling coplex. istone code q Many different aino ids in the aino terinal tails of histone proteins subject to several types of covalent odification q Pattern of odifications (histone code) affects degree of chroatin coption p Ser Arg Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. Aino terinal tail p Globular doain Ser 1 1 Arg 20 p Ser 20 Arg 3 2A p Ser B 4 (c) Repleent with variant histones

10 DNA Methylation n DNA ethylase atthes ethyl groups n Coon in soe eukaryotes but not all n In aals, % of DNA is ethylated n Usually inhibits transcription n CpG islands near prooters in vertebrates and plants q Cytosine and guanine connected by phosphodiester bonds q Unethylated CpG islands are correlated with tive genes q Repressed genes contain ethylated CpG islands Methylation can inhibit transcription two ways: 1. The Methylation of CpG islands ay prevent an tivator fro binding to an enhancer eleent 2. Converting chroatin fro an open to a closed conforation q Methyl-CpG-binding proteins bind to ethylated sequences and recruit proteins that condense the chroatin Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. Many genes are flanked by nucleosoe-free regions (NFR) and well-positioned nucleosoes. NFR NFR Regulation of RNA Processing and Translation in Eukaryotes Enhancer Transcriptional start site Activator Transcriptional terination site 2 +2 n Unlike bteria, gene expression is coonly regulated at the level of RNA processing and translation Enhancer istone etyltransferase 2 Chroatinreodeling coplex +2 Preinitiation coplex 2 1 Deetylated histones Pre- pen coplex Evicted histone proteins Chaperone n Added benefits include q Produce ore than one transcript fro a single gene (gene encodes 2 or ore polypeptides) q Faster regulation hieved by controlling steps after RNA transcript ade 40

11 Alternative splicing of pre-s n In eukaryotes, a pre- transcript is processed before it becoes a ature n When a pre- has ultiple introns and exons, splicing ay occur in ore than one way n Alternative splicing causes s to contain different patterns of exons. n Allows sae gene to ake different proteins q At different stages of developent q In different cell types q In response to a change in the environental conditions Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. α-tropoyosin pre- Exons Introns Pre- Splicing Splicing in sooth in striated uscle cells uscle cells n Linear order of exons is aintained in both alternates n Products usually have siilar functions, because ost of the aino id sequence will be the sae n Alternative splicing produces proteins with specialized charteristics n Advantage of alternative splicing: q Different polypeptides fro a single gene Constitutive exons Alternative exons Introns 41 q Bigger proteoe with the sae size genoe 42 MicroRNAs n irnas are sall RNA olecules that silence the expression of pre-existing s q Forerly known as sall or short interfering RNA (sirna) n Widely found in anials and plants n Iportant echanis of silencing n Effect also called RNA interference (RNAi) 43 n Synthesized as pre-irna n Cut by dicer to release irna n Associates with cellular proteins to becoe RNA-induced silencing coplex (RISC) n Upon binding, either q degraded q r, RISC inhibits translation Pre-iRNA Cellular Copyright The McGraw-ill Copanies, Inc. Perission required for reproduction or display. n Either way, is The is Translation is degraded (high inhibited (low silenced copleentarity copleentarity 44 sirna of bases) R The double-stranded region of a pre-irna (shown here) or a pre-sirna (not shown) is cut by dicer and releases a 22-bp RNA. The double-stranded irna or sirna associates with proteins to for RISC. ne of the RNA strands is degraded. RISC binds to a cellular due to copleentarity with the irna or sirna within RISC. of bases). irna RISC RISC 11