Presentation on theme: "Regulation of Gene Expression I"— Presentation transcript:
1 Regulation of Gene Expression I 4/15/2017Scotty MerrellDepartment of Microbiology and ImmunologyB4140Regulation of Gene Expression I
2 QUESTIONS 1. Why does the expression of genes need to be regulated? 4/15/2017QUESTIONS1. Why does the expression of genes need tobe regulated?2. Why is it important to study gene regulation?3. How is the expression of genes regulated?4. How do we study gene regulation?
3 Bacteria experience different conditions depending on environment 4/15/2017Pathogenic bacteria:External reservoir HostInfection site #1 Infection site #2
4 QUESTIONS 1. Why does the expression of genes need to be regulated? 4/15/2017QUESTIONS1. Why does the expression of genes need tobe regulated?2. Why is it important to study gene regulation?3. How is the expression of genes regulated?4. How do we study gene regulation?
5 Pathogenic bacteria produce virulence factors when they sense they are inside of a host4/15/2017ICDDR,BVibrio cholerae, the cause of cholera, produces toxin insideof the host. Understanding regulation of expression of this toxinis a means of understanding ways to prevent its production.
6 QUESTIONS 1. Why does the expression of genes need to be regulated? 4/15/2017QUESTIONS1. Why does the expression of genes need tobe regulated?2. Why is it important to study gene regulation?3. How is the expression of genes regulated?4. How do we study gene regulation?
10 RNA polymerase-promoter interactions 4/15/2017RNA polymerase-promoter interactionsSome promoters contain UP elements that stimulate transcriptionthrough direct interaction with the C-terminal domains of the subunits of the RNA polymerase
11 Arrangement of a subunits on UP elements 4/15/2017Promoter with a full UP element containing two consensus subsites.Promoter with an UP element containing only a consensus proximal subsite.Promoter with an UP element containing only a consensus distal subsite.
12 Genes come in two main flavors: 4/15/2017Genes come in two main flavors:Constitutively expressed (transcription initiationis not regulated by accessory proteins)Regulated (transcription initiationis regulated by accessory proteins)a. Negatively Regulated--Repressor Proteinb. Positively Regulated--Activator Protein
13 Mechanisms of Regulation of Transcription Initiation: Negative Regulation4/15/2017RNA Polymerase
14 Mechanisms of Regulation of Transcription Initiation: Negative Regulation4/15/2017RepressorCo-repressorInactivatorRepressorRepressor
15 a model for negative regulation The lac operona model for negative regulation4/15/2017A bacterium's prime source of food is glucose, since it doesnot have to be modified to enter the respiratory pathway. Soif both glucose and lactose are around, the bacterium wants toturn off lactose metabolism in favor of glucose metabolism.There are sites upstream of the lac genes that respond toglucose concentration.This assortment of genes and their regulatory regions is calledthe lac operon.
19 The lac promoter and operator regions 4/15/2017
20 prevents transcription The Lac Repressor is constitutively expressed4/15/2017Lac Repressor(monomer)(tetramer)Repressor bindingprevents transcription
21 the cell and binds to the Lac repressor, inducing a conformational When lactose is present, it acts as an inducer of the operon. It entersthe cell and binds to the Lac repressor, inducing a conformationalchange that allows the repressor to fall off the DNA. Now the RNApolymerase is free to move along the DNA and RNA can be made fromthe three genes. Lactose can now be metabolized.4/15/2017Remember, therepressor actsas a tetramer
22 When the inducer (lactose) is removed, the repressor returns to its original conformation and binds to the DNA, so that RNA polymerasecan no longer get past the promoter to begin transcription. No RNA andno protein are made.4/15/2017Remember, therepressor actsas a tetramer
23 How to identify the regulatory elements? 4/15/2017How to identify the regulatory elements?1. Mutation in the regulatory circuit may either abolishexpression of the operon or cause it to occur withoutresponding to regulation.2. Two classes of mutants:A. Uninducible mutants: mutants cannot be expressed at all.B. Constitutive mutants: mutants continuously expressgenes that do not respond to regulation.3. Operator (lacO): cis-acting elementRepressor (lacI): trans-acting element
24 Definitions: cis-configuration: description of two sites on the same 4/15/2017cis-configuration: description of two sites on the sameDNA molecule (chromosome)or adjacent sites.cis dominance: the ability of a gene to affect genesnext to it on the same DNA molecule(chromosome), regardless of the natureof the trans copy. Such mutations exerttheir effect, not because of alteredproducts they encode, but because of a physicalblockage or inhibition of RNA transcription.trans-configuration:description of two sites on differentDNA molecules (chromosomes)or non-contiguous sites.
25 Constitutive mutants: do not respond to regulation. 4/15/2017Would this be a cis-dominant or recessive mutation?
26 Constitutive mutants can be recessive 4/15/2017Constitutive mutants can be recessive
27 Constitutive mutants can also be dominant if the mutant allele produces a “bad” subunit, which is not only itself unable to bind to operator DNA, but is also able to act as part of a tetramer to prevent any “good” (wild type LacI) subunits from binding.4/15/2017PilacI-POlacZlacYlacAXmRNAmRNAet al.mRNAlacI+
28 Think about how you could determine whether a mutation was dominant or 4/15/2017Think about how you could determinewhether a mutation was dominant orrecessive.
29 Questions about negative 4/15/2017Questions about negativeRegulation of lac ?
30 Mechanisms of Regulation of Transcription Initiation: Positive Regulation4/15/2017RNA Polymerase
31 Mechanisms of Regulation of Transcription Initiation: Positive Regulation4/15/2017RNA PolymeraseActivator
32 a model for positive regulation The lac operona model for positive regulation4/15/2017When levels of glucose (a catabolite) in the cell are high, a molecule called cyclic AMP is inhibited from forming. So when glucose levels drop, more cAMP forms. cAMP binds to a protein called CAP (catabolite activator protein), which is then activated to bind to the CAP binding site. This activates transcription, perhaps by increasing the affinity of the site for RNA polymerase. This phenomenon is called catabolite repression, a misnomer since it involves activation, but understandable since when it was named, it seemed that the presence of glucose repressed all the other sugar metabolism operons.
33 CAP --- a positive regulator 4/15/2017CAP --- a positive regulator1. Catabolite repression: the decreased expression of many bacterial operons that results from addition of glucose. Also known as “glucose effect” or “glucose repression”.2. E. coli catabolite gene activator protein (CAP; also known as CRP, the cAMP receptor protein).3. CAP-cAMP activates more than 100 different promoters, including promoters required for utilization of alternative carbohydrate carbon sources such as lactose, galactose, arabinose, and maltose.
35 How does glucose reduce cAMP level? 4/15/2017How does glucose reduce cAMP level?PTSGlucoseGlucose-6-PIIAGlc-PIIAGlcOUTINPTS - phosphoenolpyruvate-dependent carbohydratephosphotransferase systemIIAGlc - glucose-specific IIA protein, one of theenzymes involved in glucose transport.1. IIAGlc-P activates adenylate cyclase.2. Glucose decreases IIAGlc-P level,thus reducing cAMP production.3. Glucose also reduces CAP level:crp gene is auto-regulated byCAP-cAMP.
36 Activation of expression of the lac operon 4/15/2017
37 E. coli CAP (CRP) --- 209 amino acids 4/15/2017E. coli CAP (CRP) amino acidsDNA-bindingHelix-turn-helixDimerization and cAMP-binding1-139NH2-AR1-COOHHis19His21Glu96Lys101AR2
38 Transcription activation by CAP at class I CAP-dependent promoters 4/15/2017(-62)Transcription activation:Interaction between the AR1 of the downstream CAP subunit and one copy of aCTD.The AR1-aCTD interaction facilitates the binding of aCTD to the DNA downstream of CAP.Possibly, interaction between same copy of aCTD and the s70 bound at the –35 element.4. The interaction between the second aCTD and CAP is unclear.The result: increasing the affinity of RNAP for promoter DNA, resulting in anincrease in the binding constant KB, for the formation of the RNAP-promoter closed complex
39 Transcription activation by CAP at class I CAP-dependent promoters (cont.)4/15/2017(-103, -93, -83, or –72)Transcription activation:Possibly, the second copy of aCTD may interact with the DNA downstream of CAP, andmay interact with the s70 bound at the –35 element.Results: increasing the affinity of RNAP for promoter DNA, resulting in anincrease in the binding constant KB, for the formation of the RNAP-promoter closed complex
40 Transcription activation by CAP at class II CAP-dependent promoters (cont.)4/15/2017(-42)Transcription activation:Interaction between the AR1 of the upstream CAP subunit and one copy of aCTD(either aCTDI or aCTDII, but preferentially aCTDI). The AR1-aCTDinteraction facilitates the binding of aCTD to the DNA upstream of CAP.Results: increase in the binding constant KB, for the formation of the RNAP-promoterclosed complexInteraction between the AR2 of the downstream CAP subunit and aNTDI.Result: increase the rate constant, kf, for isomerization of closed complex to open complex.
41 Transcription activation by CAP at class III CAP-dependent promoters 4/15/2017(-103 or –93)(-62)Transcription activation:Each CAP dimer functions through a class I mechanism with AR1 of the downstream subunit of each CAP dimer interacting with one copy of aCTDResults: synergistic transcription activation
42 Transcription activation by CAP at class III CAP-dependent promoters (cont.)4/15/2017(-103, -93, or -83)(-42)Transcription activation:The upstream CAP dimer functions by a class I mechanism, with AR1 of the downstream subunit interacting with one copy of aCTD; the downstream CAP dimer functions by a class II mechanism, with AR1 and AR2 interacting with the other copy of aCTD and aNTD, respectively.Results: synergistic transcription activation
43 No lactose inside the cells! 4/15/2017(a) Glucose present (cAMP low); no lactose;(b) Glucose present (cAMP low); lactose presentlacIPiPOlacYlacZlacARepressormonomertetramermRNAXNo lactose inside the cells!(inducer exclusion)!RepressormonomertetramermRNAInducerHigh levelof mRNAXInactiverepressorHigh(c) No glucose (cAMP high); lactose presentcAMPCAPGlucose effect onthe E. coli lac operon
44 No lactose inside the cells! 4/15/2017(a) Glucose present (cAMP low); no lactose;(b) Glucose present (cAMP low); lactose presentlacIPiPOlacYlacZlacARepressormonomertetramermRNAXNo lactose inside the cells!(inducer exclusion)!RepressormonomertetramermRNAInducerHigh levelof mRNAXInactiverepressorHigh(c) No glucose (cAMP high); lactose presentcAMPCAPGlucose effect onthe E. coli lac operon
45 Inducer exclusion: How does it work? 4/15/2017Uptake of glucose dephosphorylates enzyme IIglc.Dephosphorylated enzyme IIglc binds to and inhibits lactose permease.Inhibition of lactose permease prevents lactose from entering the cell.Hence, the term inducer exclusion.
46 Questions about positive regulation 4/15/2017Questions about positive regulationof the lac operon?
47 Dual positive and negative control of transcription initiation: 4/15/2017Dual positive and negative controlof transcription initiation:the E. coli ara operon
48 The E. coli L-arabinose operon 4/15/2017The E. coli L-arabinose operon++
49 AraC exists in two states 4/15/2017ArabinoseP1P2AntiactivatorActivatorArabinose
50 AraC acts as a positive or negative regulator based on its conformational state and binding affinity for various sites in the two promoter regions.4/15/2017AraC encodes the regulatorAraO1 and AraO2 encode operatorsCAP is a CAP binding siteAraI is an additional regulatory regionAraBAD are the structural genes
51 In the absence of arabinose, the P1 form of AraC binds AraO2 and AraI to prevent any P2 form from binding and activating expression--this is anti-activation, not repression!4/15/2017No arabinose+ arabinoseIn the presence of arabinsose, AraC shifts to the P2 form and bindsAraI and acts to activate transcription.If AraC concentration becomes too high, AraC will also bind to AraO1and repress its own expression.Therefore AraC is an Activator, Repressor and Anti-activator!!
52 The domain structure of one subunit of the dimeric AraC protein 4/15/2017The regulatory regions of the PC and PBAD promotersThe domain structureof one subunit of thedimeric AraC protein
53 The PC and PBAD Regions in the presence or absence of arabinose 4/15/2017The PC and PBAD Regions in the presenceor absence of arabinose+ L-arabinose
54 Hypothetical model of the activation of the PBAD promoter 4/15/2017Hypothetical model of the activation of the PBAD promoterPBAD – class II promoterPossible interactions: between the aCTD of RNAPand the CAP protein and AraC protein and DNA
55 Strategies for Understanding Regulation 4/15/2017Strategies for Understanding Regulation1. Find mutations that render the regulation uninducible or constitutive.2. Decide by performing a complementation test if the mutants are dominant orrecessive.3. If they are recessive, decide if the system is regulated by repression or byactivation. A recessive mutated activator has most likely lost function: thesystem will become uninducible. A recessive mutated repressor has also lostfunction, but now the system will show constitutive expression.4. Decide if the elements of the system act in cis or in trans to each other: arethey proteins or DNA binding sites?5. Construct a model.
56 Questions about ara regulation? 4/15/2017Questions about ara regulation?
57 A. Transcriptional control 1. Transcription initiation a) Positive 4/15/2017Regulatory mechanisms used to control gene expressionA. Transcriptional control1. Transcription initiationa) Positiveb) Negative2. Transcription terminationAttenuationB. Translational control1. Positive2. NegativeC. Post-translational control--Proteolysis
59 Transcription termination players: and sometimes the Rho (r) factor 4/15/2017Transcription termination players:Termination sequenceRNA polymeraseand sometimes the Rho (r) factorRNAPABCDXPromoterOperon of 4 genesTerminator
60 Two major types of Terminator Sequences 1. Rho-independent2. Rho-dependent4/15/2017Rho-independentterminatorRho-independentterminatorRho-dependentterminator
61 Premature termination of transcription 4/15/2017Attenuation:Premature termination of transcriptionof operons for amino acid biosynthesis(trp, his, leu, etc.)Bacterial version ofSupply and DemandRelies on coupled transcription and translation andRNA secondary structure
62 Organization of Tryptophane Biosynthesis Genes 4/15/2017Organization of Tryptophane Biosynthesis GenesEnd product of the pathwayThe trp leader mRNA encodes the LEADER PEPTIDEMetLysAlaIlePheValLeuLysGlyTrpTrpArgThrSer5’-AUGAAAGCAAUUUUCGUACUGAAAGGUUGGUGGCGCACUUCCUCCCAUAGACUAACGAAAUGCGUACCACUUAUGUGACGGGCAAAGAGCCCGCCUAAUGAGCGGGCUUUUUUUUGAACAAAAUUAGAGA-3’1324
63 mRNA forms secondary structures 4/15/20171234Pre-emptor2 and 3 form thePre-emptor, which preventsTerminator formation3 and 4 form aRho-independentterminatorTwo possible alternative structures can form2 is complementary to 1 and 33 is complementary to 2 and 4Adapted from