Liam.Keegan@ceitec.muni.cz Lewin, GENES XI Chapter 26 Operons, prokaryotic gene regulation and yeast GAL4 Lewin's GENES XII (PDF) https://pdfroom.com/books/lewins-qenes-xii/Wx5aDYKI2BJ biotecher.ir https://biotecher.ir/wp-content/uploads/2018/06/bio... Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Mentioned in splicing lecture • [HTML] Intron-rich dinoflagellate genomes driven by Introner transposable elements of unprecedented diversity • SW Roy, L Gozashti, BA Bowser, BN Weinstein... - Current Biology, 2023 - Elsevier Spliceosomal introns, which interrupt nuclear genes, are ubiquitous features of eukaryotic nuclear genes. 1 Spliceosomal intron evolution is complex, with different lineages ranging ... • Save Cite Cited by 1 Related articles All 4 versions Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com LEWIN'S Background image © Photo Researchers, Inc. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.2 Operons are Structural Gene Clusters that Are Coordinately Controlled • Genes coding for proteins that function in the same pathway may be located adjacent to one another and controlled as a single unit that is transcribed into a polycistronic mRNA Figure 26.05: The lac operon occupies ~6000 bp of DNA. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.1 Introduction In negative regulation, a repressor protein binds to an operator to prevent a gene from being expressed. In positive regulation, a transcription factor is required to bind at the promoter in order to enable RNA polymerase to initiate transcription. cis-acting operator/promoter precedes structural gene(s) Promoter operator Structural gene(s) Gene on: RNA polymerase initiates at promoter RNA _Protein Gene is turned of( when repressor binds to operator Repressor GENE OFF BY [ Promoter I )EFAULT Start point I vf v< v/ vf v./ v< v< v< v/ s/ v/ v/ GENE TURNED ON 3Y ACTIVATORS Factors interact with RNA polymerase RNA Figure 26.02: In negative control, a transacting repressor binds to the cis-acting operator to turn off transcription. X/X/\/ Protein Figure 26.03: In positive control, a transacting factor must bind to cis-acting site in order for RNA polymerase to initiate transcription at the promoter. Copyright © 2013 by Jones & Bartiett Learning, LLC an Ascend Learning Company www.jblearning.com 26.1 Introduction • In inducible regulation, the gene is regulated by the presence of its substrate (the inducer). This is good for catabolic pathways for adapting to new food sources, for instance to use lactose or galactose sugars to grow if there is no glucose. • In repressible regulation, the gene is regulated by the product of its enzyme pathway (the corepressor). Genes encoding enzymes in anabolic pathways to make amino acids or nucleotides or other needed products can reduce expression when there is enough product. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.3 Famous inducible genes and repressors in E. coli The lac Operon Is under Negative control by lac repressor Start point Unwinding -50 -40 -30 -20 -10 1 10 4 Promoter n» 20 30 binds RNA polymerase Operator binds repressor Figure 26.06: lac repressor and RNA polymerase bind at sites that overlap around the transcription startpoint of the lac operon. Transcription of the lacZYA operon is controlled by a repressor protein (the lac repressor) that binds to an operator that overlaps the promoter at the start of the cluster. In the absence of (3-galactosides, the lac operon is expressed only at a very low (basal) level. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com lacZ (p-galactosidase)) plate tests and liquid culture assays fo beta-gal activity units. HOCH£ ONPG hydrolysis by cells gives a soluble blue product for spectrophotometric measurement of lacZ ((3-galactosidase ) activity units Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.3 The lac Operon Is under negative transcriptional control by repressor and is inducible by galactosides • The repressor protein is a tetramer of identical subunits coded by the lacl gene. • (3-galactoside sugars, the substrates of the lac operon, are its inducer. • Addition of specific (3-galactosides induces transcription of all three genes of the lac operon. • The lac mRNA is extremely unstable; as a result, induction can be rapidly reversed. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.3 The lac Operon Is Negative Inducible Add inducer Remove inducer t Basal leve 4 6 8 10 Level of teem RNA 12 rv-in Induced level 2 4 6 8 10 Level of Ji-gaiactosidase 12 mm Figure 26.07: Addition of inducer results in rapid induction of lac mRNA, and is followed after a short lag by synthesis of the enzymes. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.4 lac Repressor Is Controlled by a Small-Molecule Inducer AAA Tctramcr binds to operator and blocks transcription Promoter/ lacZ iacY iacA Operator tad gene synthesizes repressor monomer that forms tetramer Monomer Tetramer ^^^^^^1 Figure 26.08: lac repressor maintains the lac operon in the inactive condition by binding to the operator. An inducer functions by converting the repressor protein into a form with lower operator affinity. Repressor has two binding sites, one for the operator DNA and another for the inducer. gratuitous inducer - Inducers that resemble authentic inducers of transcription, but are not substrates for the induced enzymes.(IPTG for lac repressor) Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.4 lac Repressor Is Controlled by a Small-Molecule Inducer • Repressor is inactivated by an allosteric interaction in which binding of inducer at its site changes the properties of the DNA-binding site (allosteric control). • The true inducer is allolactose, not the actual substrate of (3-galactosidase. In experiments we induce with artificial IPTG, not metabolized, a gratuitous inducer. INDUCER—* s\s\ Inducer converts lac repressor into a form with tow affinity ior operator Figure 26.09: Addition of inducer converts repressor to a form with low affinity for the operator. This allows RNA polymerase to initiate transcription. RNA polymerase binds at promoter and transcribes RNA \/\/\/\/x/\/ m RNA is translated Into all three proteins Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.5 c/s-Acting Constitutive Mutations Identify the Operator • Mutations in the operator cause constitutive expression of all three lac structural genes. • constitutive expression - A state in which a gene is expressed continuously. • These mutations are c/s-acting and affect only those genes on the contiguous stretch of DNA. • Mutations in the promoter prevent expression of lacZYA and are uninducible and c/s-acting. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.5 c/s-Acting Constitutive Mutations Identify the Operator • c/s-dominant - A site or mutation that affects the properties only of its own molecule of DNA, often indicating that a site does not code for a diffusible product. Oc means 'operator constitutive' mutation i—• ii t ■ t Repressor cannot bind * to mutant operator t : x Figure 26.10: Operator mutations are constitutive because the operator is unable to bind repressor protein. 0° operator /\/\/\s\/\ Operon is transcribed . and translated t © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.6 trans-Acting Mutations Identify the Regulator Gene Mutations in the lacl gene are trans-aci\ng and affect expression of all lacZYA clusters in the bacterium. Mutations that eliminate lacl function cause constitutive expression and are recessive (lach). Mutations in the DNA-binding site of the repressor are constitutive because the repressor cannot bind the operator, (also cis-acting) t t !acl~ gene synthesizes defective repressor that does not bind to operator Operon is transcribed /\/\S\/\/\ and translated Figure 26.11: Mutations that inactivate the lacl gene cause the operon to be constitutively expressed. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearntng.com 26.6 trans-AcWng Mutations Identify the Regulator Gene • Mutations in the inducer-binding site of the repressor prevent it from being inactivated and cause uninducibility. Super-repressor mutants. • The opposite is a dominant negative lachd repressor mutant. When mutant and wild-type subunits are present, a single lacH mutant subunit can inactivate a tetramer whose other subunits are wild-type. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.6 řrans-Acting Mutations Identify the Regulator Gene ^ lact^ mutant synthesis repressor with defective DNA-binding site J Wild-type lact gene synthesizes normal repressor One nbad" subunit poisons the tetramer; cannot bind DNA normally so operon expressed s\/\/\/\/\ Figure 26.12: A lacl-d mutant gene makes a monomer that has a damaged DNA binding. negative complementation, i.e dominant negative - This occurs when interallelic complementation allows a mutant subunit to suppress the activity of a wild-type subunit in a multimeric protein. Iachd mutations occur in the DNA-binding domain. Their effect is explained by the fact that repressor activity requires all DNA-binding domains in the tetramer to be Copyrig Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.7 lac Repressor Is a Tetramer Made of Two Dinners • A single repressor subunit can be divided into the N-terminal DNA-binding domain, a hinge, and the core of the protein. • The DNA-binding domain contains two short a-helical regions that bind the major groove of DNA. • The inducer-binding site and the regions responsible for multimerization are located in the core. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.7 lac Repressor Is a Tetramer Made of Two Dimers turn Figure 26.13: The structure of a monomer of Lac repressor identifies several independent domains. Structure from Protein Data Bank 1LBG. M. Lewis, et al., c ■ -.-7-1 iinnr\ iia-iiita nu 4. 4. t: 11 i- Copyriqht © 2013 by Jones & Bartiett Learning, LLC an Ascend Learninq Company Science 271 (1996): 1247-1254. Photo courtesy of Hongh K/ s 7 s ., , . , , , www.jblearning.com Zhan and Kathleen S. Matthews, Rice University. 26.7 lac Repressor Is a Tetramer Made of Two Dimers DNA-binding domains Monomers form a dimer by making contacts between core subdomains 1 and 2. Dimers form a tetramer by interactions between the tetramerization helices. Figure 26.15: The repressor tetramer Copyright © 2013 by Jones & Bartiett Learning, LLC an Ascend Learning Company consists of two di merS. www.jblearning.com 26.7 lac Repressor Is a Tetramer Made of Two Dimers N-terminus Sites of mutations Different types of mutations occur in different domains of the repressor protein. Figure 26.16: The locations of three type of mutations in lactose repressor are mapped on the domain StrUCtUre Of the DrOtein Copyright ©2013 by Jones&Bartlett Learning, LLC an Ascend Learning Company " ' www.jblearning.com 26.8 lac Repressor Binding to the Operator Is Regulated by an Allosteric Change in Conformation Headpieces bind successive turns in major groove • Inducer binding causes a change in repressor conformation that reduces its affinity for DNA and releases it from the operator. Inducer binding changes conformation --Inducer Figure 26.18: The inducer changes the structure of the core. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.8 lac Repressor Binding to the Operator Is Regulated by an Allosteric Change in Conformation » lac repressor protein binds to the double-stranded DNA sequence of the operator. > The operator is a palindromic sequence of 26 bp. > Each inverted repeat of the operator binds to the DNA-binding site of one repressor subunit. 'TiRNA TGTTGTGTGGAATTGAGAGCGG AT AACAATTTCAC ACA ACAACACACCTTAACACTCGCCT A T T G T T AAA G T G T G T -10 -5 +1 +5 +10 +15 +20 +25 Axis of symmetry Figure 26.17: The lac operator has a symmetrical sequence. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.9 lac Repressor Binds to Three Operators and Interacts with RNA Polymerase Each dimer in a repressor tetramer can bind an operator, so that the tetramer can bind two operators simultaneously. Full repression requires the repressor to bind to an additional operator downstream or upstream as well as to the primary operator at the lacZ promoter. Binding of repressor at the operator stimulates binding of RNA polymerase at the promoter but precludes transcription. Figure 26.21: If both dinners in a repressor tetramer bind to DNA, the DNA between the two binding sites is held in a loop. Third operator not shown. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.11 Positive regulators of transcription The lac Operon Has a Second Layer of Control: Catabolite Repression • catabolite repression - The ability of glucose to prevent the expression of a number of genes. - In bacteria this is a positive control system; in eukaryotes, it is completely different. • Catabolite repressor protein (CRP) is an activator protein that binds to a target sequence at a promoter. • CRP is or was also known as cAMP-dependent, Catabolite Activator Protein (CAP), a newer name I find clearer. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.11 The lac Operon Has a Second Layer of Control: Catabolite Repression Repressed Induced RNA polymerase O Inducer (CAMP) Figure 26.25: cAMP converts an activator protein CRP to a form that binds the promoter and assists RNA polymerase in initiating transcription. Copyright © 2013 by Jones & Bartiett Learning, LLC an Ascend Learning Company www.jblearning.com 26.11 The lac Operon Has a Second Layer of Control: Catabolite Repression of \ Glucose 1 • A dimer of CRP is activated by \ Reduced cAMP 1 a single molecule of cyclic AMP (cAMP). h J T • cAMP is controlled by the Active CRP 1 Inactive CRP level of glucose in the cell; a low glucose level allows d.h. j cAMP to be made. • CRP interacts with the C- 1 No transcription terminal domain of the a subunit of RNA polymerase to Figure 26.27: By reducing the level of activate it. cyclic AMP, glucose inhibits the transcription of operons that require CRP activity. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 27.1 Introduction Famous inducible genes and transcriptional repressors in E. coli • bacteriophage (or phage) - A bacterial virus. • lytic infection - Infection of a bacterium by a phage that ends in the destruction of the bacterium with release of progeny phage. • lysis - The death of bacteria at the end of a phage infective cycle when they burst open to release the progeny of an infecting phage (because phage enzymes disrupt the bacterium's cytoplasmic membrane or cell wall). Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 27.1 Introduction : " Bacterisr^NA LYTIC CYCLE LYSOGENY ' Phage DNA is integrated into bacterial ■ genome; bacteria live happily ever after j« Progeny phages are released from lysed bacterium • Lysogenic bacterium is • immune to further infection ■ ♦ INDUCTION Figure 27.01: Lytic development involves the reproduction of phage particles with destruction of the host bacterium. Gateway and other cloning and directed recombination systems use phage proteins and target sites like the lambda att site Phage DNA is released and enters lytic cycle Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com A bacteriophage plaques on a lawn of E. coli are clear where all cells were killed by virus infection and cell lysis (- like a coldsore). Cloudy plaques contain A lysogen cells that are immune to further lambda virus infection leading directly to cell lysis Ascend Learning Company www.jblearning.com Clear pfaque 27.9 The Lambda Repressor and Its Operators Define the Immunity Region • immunity - In phages, the ability of a prophage to prevent another phage of the same type from infecting a cell. • virulent mutations - Phage mutants that are unable to establish lysogeny. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 27.9 The Lambda Repressor and Its Operators Define the Immunity Region • Several lambdoid phages have different immunity regions. • A lysogenic phage confers immunity to further infection by any other phage with the same immunity region. RNA polymerase cannot initiate at Pm in absence of repressor Figure 27.16: In the absence of repressor, RNA polymerase initiates at the left and right promoters. rna polymerase initiates at PL RNA\/\y\/ cro rn Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Repressor dimer Y am Repressor prevents RNA polymerase from biriding PL 27.8 Lysogeny Is Maintained by the Lambda Repressor Protein • The lambda repressor, encoded by the cl gene, is required to maintain lysogeny • The lambda repressor acts at the 0L and 0R operators to block transcription of the immediate early genes. • The immediate early genes trigger a regulatory cascade; as a result, their repression prevents the lytic cycle from proceeding. monomer vlA t ~^^^PrM cl repressor gene Repressor prevents RNA polymerase from binding Pr Figure 27.15: Repressor acts at the left operator and right operator to prevent transcription of the immediate early genes (N and cro). Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Need to purify scarce gene regulator proteins drove biotechnology of protein overexpression • Take E. coli cell as a cube 1 micron on each side. A liter is a cube 10 cm on each side. What is the concentration of the lac operator? (Answer, nanomolar; about 10~9 M). 10-100 lac repressor molecules per cell and the kd for operator-binding is 1010 M or lower (tighter). • Lambda repressor was first isolated from an overproducer mutant virus. Later the repressor gene was expressed from lac or toe promoters. Also, a hybrid ribosome binding site (Shine-Dalgarno sequence), upstream of ATG gave strong translation initiation. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Sequence-specific DNA recognition is the key to differential gene regulation. Mark Ptashne with graduate students Cynthia Wohlberger, Liam Keegan, Ed Giniger at Harvard, 1982 Copyright © 2013 by Jones & Bartiett Learning, LLC an Ascend Learning Company www.jblearning.com 27.10 The DNA-Binding Form of the Lambda Repressor Is a Dimer Monomers are in equilibrium with dimers, which bind to DMA Cleavage ot monomers disturbs equilibrium, so dimers dissociate INDUCTION Cleavage T • A repressor monomer has two distinct domains. • The N-terminal domain contains the DNA-binding site. • The C-terminal domain dimerizes. • Binding to the operator requires the dimeric form so that two DNA-binding domains can contact the operator simultaneously. • Cleavage of the repressor between the two domains reduces the affinity for the operator and induces a lytic cycle. Figure 27.18: Repressor dimers bind to the operator. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 27.11 Lambda Repressor Uses a Helix-Turn-Helix Motif to Bind DNA • Each DNA-binding region in the repressor contacts a half-site in the DNA. • The DNA-binding site of the repressor includes two short a-helical regions that fit into the successive turns of the major groove of DNA (helix-turn-helix). • A DNA-binding site is a (partially) palindromic sequence of 17 bp. Figure 27.19: The operator is a 17-bp sequence with an axis of symmetry through the TACCTCTGG CGGTG AT£ AT G G AG AC C G CC ACTAT central base pair. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Lambda repressor binds as a symmetrical protein dimer to a nearly symmetrical 17 bp DNA sequence. Symmetric repressor site 5'tatcaccg c*cg g tg ata atagtggcggccactat5 ■4- ^^^^^^^^^^^^^^^^^^ ♦ FIGURE 16-11 Binding of a protein with a helix-turn-helix domain to DNA. The protein, as is typically the case, binds as a dimer, and the two subunits are indicated by the shaded circles. The helix-turn-helix motif on each monomer is indicated; the "recognition helix" is labeled R. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Recognition helix amino acid side-chains make many sequence-specific contacts to base pairs. 5'tatcaccgccggtgata atagtggcggccactat5' <- 5' P 3' OH FIGURE 16-12 Hydrogen bonds between A repressor and base pairs in the major groove of its operator. Diagram of the represser-operator complex, showing hydrogen bonds (in dotted lines) between amino add side chains and bases in the consensus half-site. Only the relevant amino acid side chains are shown. In addition to Gln44 and 5er45 in the recognition helix, AsnSS in the loop following the recognition helix also makes contact with a specific base. Furthermore (and unusual to this case, see later in the text) Lys4 in the N-terminal arm of the protein makes a contact in the major groove on the opposite face of the DNA helix. Gln33 contacts the backbone. (Source: Redrawn from Jordan, S. and Pa bo, C. Science 242: 896, Fig. 3 BO www.jblearning.com Example of an amino acid contact that can discriminate between bases in a binding site. minor groove toDNA- H' N N- H C N-H H / N \ H P □ C-CH2-CH2-C-C^ -'■ protein * I H H II glutamine major groove The position of the glutamine is fixed when repressor binds. Glutamine side chain needs to find the hydrogen donor and acceptor sites in just the right places. It cannot reach the paired base on the other strand. It cannot reach the other bases on the same strand. A Genetic Switch, 3rd edition, 2004 © CoEd Spring Harbor Laboratory Press Chapter 2, Figure 12 Only an A base meets the criteria. 3 by Jones & Barttett Learning, LLC an Ascend Learning Company www.jblearning.com Wide major groove means DNA sequence can be recognized by DNA-binding proteins. major groove major groove FIGURE 6-10 Chemical groups exposed in the major and minor grooves from the edges of the base pairs. The letters in red identify hydrogen bond acceptors (A), hydrogen bond donors (D), nonpolar hydrogens (H), and methyl groups (M). minor groove minor groove ■any :om 27.12 Lambda Repressor Dimers Bind Cooperatively to the Operator • Repressor binding to one operator increases the affinity for binding a second repressor dimer to the adjacent operator. • The affinity is 10 x greater for 0L1 and 0R1 than other operators, so they are bound first. • Cooperativity allows repressor to bind the 0L2/0R2 sites at lower concentrations. Figure 27.25: When two lambda repressor dimers bind cooperatively, each of the subunits of one dimer contacts a subunit in the other dimer. ) 2013 by Jones & Bartiett Learning, LLC an Ascend Learning Company www.jblearning.com 27.13 Lambda Repressor Maintains an Autoregulatory Circuit • The DNA-binding region of repressor at 0R2 contacts RNA polymerase and stabilizes its binding to PRM. • This is the basis for the autoregulatory control of repressor maintenance. • Repressor binding at 0L blocks transcription of gene N from PL. ht © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Figure 27.26: Positive control mutations identify a small region at helix-2 that interacts directly with RNA polymerase. 27.13 Lambda Repressor Maintains an Autoregulatory Circuit cypL Repressor prevents RNA polymerase from binding PL N mRNA s\/\ LYSOGENY repressor di trior repressor monomer t r /\/\X\fy cl mRNA t cl repressor gene t£B> R;' R RNA polymerase Repressor prevents binds PRM and RNA polymerase transcribes cl trom binding PR LYTIC CYCLE RNA polymerase cannot initiate at PRM in absence of repressor RNA polymerase initiates at PR RNA polymerase initiates at PL I cro mRNA S\/\ Figure 27.27: Lysogeny is maintained by an autoregulatory circuit. Repressor binding at 0R blocks transcription of cro, but also is required for transcription of cl. Repressor binding to the operators therefore simultaneously blocks entry to the lytic cycle and promotes its own synthesis. Copyright © 2013 by Jones & Bartiett Learning, LLC an Ascend Learning Company www.jblearning.com 27.14 Cooperative Interactions Increase the Sensitivity of Regulation • Repressor dimers bound at 0L1 and 0L2 interact with dimers bound at 0R1 and 0R2 to form octamers. • These cooperative interactions increase the sensitivity of regulation. State of dgene: OFF ct Or3 Or2 Or1 cm Figure 27.29: OI3 and Or3 are brought into proximity by formation of the repressor octamer. Copyright © 2013 by Jones & Bartiett Learning, LLC an Ascend Learning Company www.jblearning.com 27.11 Lambda Repressor Uses a Helix-Turn-Helix Motif to Bind DNA The amino acid sequence of the recognition helix makes contacts with particular bases in the operator sequence that it recognizes. Cro - 0^3 REPRESSOR ■ Or1 Arm TACCTCTG ATGGAGACC TATCTCTT ATAGGGAAC Figure 27.22: Two proteins that use the two-helix arrangement to contact DNA recognize lambda operators with affinities determined by helix-3. right © 2013 by Jones & Barttett Learning, LLC an Ascend Learning Company www.jblearning.com Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.3 Famous inducible genes and repressors in E. coli The trp Operon Is under Negative control by trp repressor and by transcriptional attenuation Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com gal aroH 26.12 The trp Operon Is a Repressible Operon with Three Transcription Units The trp operon is negatively controlled by the level of its product, the amino acid tryptophan (autoregulation). The amino acid tryptophan activates an inactive repressor encoded by trpR. A repressor (or activator) will act on all loci that have a copy of its target operator sequence. Startpoint trpR lac — Promotern Operator locations* Figure 26.32: Operators may lie at various positions relative to the promoter. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Transcription attenuation in the leader of the E. coli trp operon Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.13 The trp Operon Is Also Controlled by Attenuation An attenuator (intrinsic terminator) is located between the promoter and the first gene of the trp cluster. The absence of Trp-tRNA suppresses termination and results in a 10x increase in transcription. TRANSCRIPTION OF LEADER REGION Promoter Pause Attenuator trpE Polymerase initiates Polymerase pauses TRYPTOPHAN ABSENT: TRANSCRIPTION CONTINUES INTO OPERON Polymerase elongates Translation initiates J?*si£&>- Promoter ■ * Terminator 1 terminator 2 Figure 26.33: Termination can be controlled via changes in RNA secondary structure that are determined by ribosome movement. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.14 Attenuation Can Be Controlled by Translation • The leader region of the trp operon has a 14-codon open reading frame that includes two codons for tryptophan. • The structure of RNA at the attenuator depends on whether this reading frame is translated. • In the presence of Trp-tRNA, the leader is translated to a leader peptide, and the attenuator is able to form the hairpin that causes termination. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.14 Attenuation Can Be Controlled by Translation Protein Gene Indole-glycerol Tryptophan synthetase A n t h ran i I ate sy n th etase sy nth etase B cha i n A cha i n trpE trpD trpC trpB trpA t V Control region Promoter Operator Leader Attenuator PPPNJJEGMAGCMUUUUCGUA^ 26 Leader peptide terminator M61 Lys Ala lie Phe Va! L&u LyS Gly Trp Trp Arg Thr Sef ^ G-Cnlch hairpin/ U-rich single strand Figure 26.35: The trp operon has a short sequence coding for a leader peptide that is located between the operator and the attenuator. Copyright © 2013 by Jones & Bartiett Learning, LLC an Ascend Learning Company www.jblearning.com 26.14 Attenuation Can Be Controlled by Translation TRYPTOPHAN ABSENT IAJUUUUUU Regions 3 and 4 ALTERNATIVE STRUCTURES pair to form the Region 2 is complementary to 1 and 3 terminator hairpin Region 3 is complementary to 2 and 4 Regions 2 and 3 pair; terminator region is single stranded Figure 26.36: The trp leader region can exist in alternative base-paired conformations. iuuuu Ribosome halts at Trp codons TRYPTOPHAN PRESENT Ribosome advances Tr0 ugg ug g cg a ac u u ccug aa ac ggg c ag u Ribosome movement disrupts 2:3 pairing 3:4 pairing forms terminator hairpin Figure 26.37: The alternatives for RNA polymerase at the attenuator depend on the location of the ribosome. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.14 Attenuation Can Be Controlled by Translation • In the absence of Trp-tRNA, the ribosome stalls at the tryptophan codons and an alternative secondary structure prevents formation of the hairpin, so that transcription continues. TRYPTOPHAN PRESENT Figure 26.38: In the presence of tryptophan tRNA, ribosomes translate the leader peptide and are released. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Mechanism of transcription activation of the yeast GAL genes; a model for eukaryotic transcriptional gene regulation Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com A classical model of inducible gene expression in a eukaryote. • GAL1 galactokinase (mutant allele is gall) • GAL2 galactose permease • GAL7 galactose uridyl transferase • GAL10 galactose-glucose epimerase • GAL4 positive regulator (mutant is gal4) • GAL80 negative regulator (mutant is gal80) Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Yeast GAL genes are positively regulated at the level of transcription activation. • A gal4, gal80 double mutant fails to induce expression of galactose enzymes. (gal4 mutation is epistatic to gal80) • Interpretation is that GAL4 targets the structural genes to activate transcription. • GAL80 interacts with GAL4 to control its activity. Copyright © 2013 by Jones & Bartiett Learning, LLC an Ascend Learning Company www.jblearning.com Mechanism of transcription activation of the yeast GAL genes. Po* Laboratory Press, Chapter 2. Figure 5. Gal4 GalSO binding (851-881) i ARII (768-881) C ARl (148-196) Dimerization (66-94) DNA binding (1-65). Genes and Signals, 2002 by Cold Spring Harbor Laboratory Press. Chapter 2. Figure 4. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Current understanding of yeast GAL gene activation {a) Galactose absent (b) Galactose present Gal3 ■TYjl GalflOlsboundby Gat3;Gal4 Qaigg \JLf^ binds lo UASe and activates G al &Q fr~-~4»~^ transcription. Activation domain Gal* is bound by Gaiao and is Gal4 \*f%/ unable lo bind UASG- homodimer V V Gal4 GAL genes homodimer 1 . DNA-binding domain J _<*AL genes_ No transcription -,-■ !—- Transcription UAS0 UA5q GAL1-GAL80 complex DOES bind UASG DNA (a is wrong on this). GAL3 is the galactose sensor for GAL gene induction GAL3 is closely related to GAL1, also binds galactose and ATP GAL1 itself acts instead of GAL3 to bind GAL80 in Kluveromyces lactis, the whey (skimmed milk) yeast, which hydrolyses lactose and then converts the galactose to glucose Copyright © 2013 by Jones & Bartiett Learning, LLC an Ascend Learning Company www.jblearning.com Eukaryotic gene activators recruit RNA polymerase to the promoter. • Activating regions associate with mediator which in turn associates with RNA polymerase. • Screen for yeast mutants with increased activation by GAL4(1-100) identified a potentiator mutant in GAL11, a component of Mediator. • Galll when fused to LexA activates transcription. This is an activator bypass experiment. • Results favour the idea that GAL4 recruits polymerase to the promoter by helping it bind as CAP/CRP does in E. coli. Genes and Signals. ■ 2W2 by Cold Spring Harbor Laboratory Press, Chapter 2, Figure 13. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com DNA looping is the simplest explanation for GAL4 action. I >NA lcx>|) PATAA Figure 6.22 DNA looping Transcription factors bound at distant enhancers are able to interact with general transcription factors at the promoter because the intervening DNA can form loops. There is therefore no fundamental difference between the action of transcription factors bound to DNA just upstream of the promoter and to distant enhancers. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Uses of GAL4 Number 1: The yeast two hybrid system for identification of interacting proteins. b rag™ gene () -m i j oft ■ sb Igene Can be used on a genomic scale to test all proteins against all others. Target protein fused to DNA-binding domain in cells of one mating type mated to a library of activating region fusion proteins. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Uses of GAL4 Number 2: GAL4 activates transcription from UASG in Drosophila. Widely used for targeted protein expression in Drosophila (GAL4-UAS two-component system). o: GALA on QI Acne X ' 1 off tissue specific •ntiancer UASc fly 1 fly 2 I fly 3 I QAL4 * > on CL t gcneX tissue specif \«fe3r embryonic oD NA — uas - Transcri prj on of ey-vfess in ant ennal, leg and \vin g irn agin gal discs B D Induction of ectopic eyes by GAL4-targeted expression of the eyeless gene in Drosophila Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com The GAL4-UAS binary system is used to map brain neurons involved in memories and behaviours and to target gene expression there. • rutabaga mutant flies lack an adenlyl-cyclase required for synaptic plasticity and cannot learn a variety of training tasks. • A UAS-RUTABAGA construct was expressed under the control of different GAL4 drivers in a rutabaga mutant fly to see which brain cells are needed to learn particular tasks. • Mushroom body cells (somewhat similar to mammalian hippocampus) need RUTABAGA protein at synapses to learn to avoid particular odours and central complex cells need it for visual learning. Drosophila connectome is all the neuron types and pathways in the whole CNS Copyright © 2013 by Jones & Barttett Learning, LLC an Ascend Learning Company www.jblearning.com Liam.Keegan@ceitec.muni.cz Lewin, GENES XI Chapter 26 Operons, prokaryotic gene regulation and yeast GAL4 Lewin's GENES XII (PDF) https://pdfroom.com/books/lewins-qenes-xii/Wx5aDYKI2BJ biotecher.ir https://biotecher.ir/wp-content/uploads/2018/06/bio... Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Showing that GAL4 binds the GAL UAS Methods for identifying and characterising sequence-specific DNA-binding proteins. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com DNase I protection assay to precisely define binding sites of sequence-specific DNA binding proteins and to measure DNA- binding affinity, (DNase I Footprinting) The GAL4-binding site is CGG-N(11)-CCG. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com The GAL4 DNA-binding domain has a Zn2Cys6 binuclear cluster structure found in over 60 proteins but restricted to fungi. DNA sita CGG(IM:j)CCG CGG(Ni„)CCG CGGfNr.)CCG CGG(N-0CCG CGGftl.JCGG {CGGf AGGF TGG) r gt (Hi,) COG a2 14 15 14 16 19 14 Since GAL4 has a type of DNA-binding domain not seen in animals we will discuss DNA sequence recognition and more common types of DNA-binding domains tomon Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Deletion analysis of yeast GAL1 upstream region defined an Upstream Activating Site (UASG) and Migl site. a UASq 1 2 3 275 bp XT Mig1 | GAL1 D Four copies of a nearly symmetric 17 bp GAL4 site. Migl protein mediates glucose repression of GAL1 Migl recruits Ssn6 and Tupl Migl also binds GAL4 promoter and lowers GAL4 protein levels Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Molecular Genetics 3 Lecture 5 4.10.11 Summary RNA polymerases and RNA pol II promoters. TAFs Transcriptional regulation in yeast. Uses of GAL4 Copyright © 2013 by Jones & Bartiett Learning, LLC an Ascend Learning Company www.jblearning.com Condensation and decondensation of chromatin in the cell cycle Relaxed jig lag Compjti Figure Ö Representation of the stages in folding of a nudeosomal chain {kit) via the zig-zag ribbon into the helical ribbon (right) conformation.To avoid confusion, the DNA is shown only in places, and some underlying nucleosomes have been shaded. Chromatin condenses during mitosis and decondenses in interphase when most genes are expressed. Histone deacetylases (HDACs) facilitate chromatin condensation and contribute to gene repression (Hdal, Rpd3). Histone acetyl transferases (HATs), like GCN5 in the SAGA complex, acetylate histone tails and contribute to chromatin decondensation and gene activation. Nucleosome remodelling complexes (Swi/Snf complex), can remove nucleosomes from a promoter region or deposit them there. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Imposing a requirement for nucleosome modifiers for GAL1 activation. Neither GCN5 histone acetyltransferase nor the nucleosome remodelling complex Swi/Snf complex are required for GAL1 activation nor to activate 95% of yeast genes. Nevertheless GAL4 recruits these activities to the promoter and they become necessary if GAL4 sites are weakened or when chromosomes condense for mitosis. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Unusual chromosomes in Saccharomyces cerevisiae • The nuclear membrane was seen only after the electron microscope was invented. The nuclear membrane does not break down at mitosis. • The genome is small and divided among 16 chromosomes. 5. cerevisiae lacks a Histone protein 1 that binds the linker DNA between nucleosomes and causes them to condense more. Cell division takes place without formation of visible metaphase chromosomes. • The fission yeast Schizosaccharomyces pombe has fewer chromosomes and these become visible at metaphase. It is quite different from budding yeast in DNA sequence and was promoted as being more like mammals by Paul Nurse and others who used it to study the cell cycle, ("Schizophrenic pombe"). Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.15 Stringent Control by Stable RNA Transcription • Poor growth conditions cause bacteria to produce the small-molecule regulators (p)ppGpp. • Stringent response - The ability of a bacterium to shut down synthesis of ribosomes and tRNA in a poor growth medium. • The trigger for the reaction is the entry of uncharged tRNA into the ribosomal A site. • (p)ppGpp competes with ATP during formation of the open complex during transcription initiation by RNA polymerase and inhibits the reaction. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.15 Stringent Control by Stable RNA Transcription Rel A (p)ppGpp synthesis = Stringent factor Major Pathway pppGp GDP) + - Minor Pathway ppGp Spot I GDP Figure 26.40: Stringent factor catalyzes the synthesis of pppGpp and ppGpp; ribosomal proteins can dephosphorylate pppGpp to ppGpp. Relaxed mutants - In E. co//, these do not display the stringent response to starvation for amino acids (or other nutritional deprivation). Stringent factor - The protein RelA, which is associated with ribosomes. It synthesizes ppGpp and pppGpp when an uncharged tRNA enters the ribosome. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.16 r-Protein Synthesis Is Controlled by Autoregulation • Translation of an r-protein operon can be controlled by a product of the operon that binds to a site on the polycistronic mRNA. When rRNA is available, the r-proteins associate with it. Translation of mRNA continues. When no rRNA is avail ale, r-proteins accumulate. An r-protein binds to mRNA and prevents translation. r-proteins Figure 26.42: Translation of the r-protein operons is autogenously controlled and responds to the level of rRNA. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.10 The Operator Competes with Low-Affinity Sites to Bind Repressor • Proteins that have a high affinity for a specific DNA sequence also have a low affinity for other DNA sequences. • Every base pair in the bacterial genome is the start of a low-affinity binding site for repressor. DNA Repressor Repressor + inducer Operator 2x1013 2x 1010 Other DNA 2x 1Ü6 2X106 Specificity 107 Operators bound 96% 3% Operon is: repressed induced Figure 26.23: lac repressor binds strongly and specifically to its operator, but is released by inducer. All equilibrium constants are in M-l. Copyright © 2013 by Jones & Bartiett Learning, LLC an Ascend Learning Company www.jblearning.com 26.10 The Operator Competes with Low-Affinity Sites to Bind Repressor MAINTAINING REPRESSION Repressor is bound \ at operator \ Excess repressor bound elsewhere on DNA I Inducer + INDUCTION T Repressor is released from operator, and all repressors are bound at random sites on DNA Remove inducer ESTABLISHING REPRESSION Repressor returns to active form and moves from random site to operator by sliding or by direct displacement Figure 26.24: Virtually all the repressor in the cell is bound to DNA. The large number of low-affinity sites ensures that all repressor protein is bound to DNA. Repressor binds to the operator by moving from a low-affinity site rather than by equilibrating from solution. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.10 The Operator Competes with Low-Affinity Sites to Bind Repressor • In the absence of inducer, the operator has an affinity for repressor that is 107 times that of a low-affinity site. • The level of 10 repressor tetramers per cell ensures that the operator is bound by repressor 96% of the time. • Induction reduces the affinity for the operator to 104 times that of low-affinity sites, so that operator is bound only 3% of the time. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Q to LU tr <- LU tr NEGATIVE CONTROL i i Repressor REPRESSED I Ina Inactive ^ repressor Inducer INDUCED I Inactive ' Active repressor repressor Corepressor INDUCED REPRESSED POSITIVE CONTROL RNA polymerase T Inactive Active activator Q activator Inducer REPRESSED INDUCED I I Active activator r inactive activator Corepressor INDUCED REPRESSED -- 26.1 Introduction negative control of transcription by a repressor removed by an inducer (lac repressor + IPTG), positive control of transcription by activator and inducer (CAP + cAMP), (also possible, lower half, negative control of transcription by a repressor and corepressor and positive control of transcription by having an activator that is removed) Figure 26.04: Regulatory circuits can be designed from all possible combinations of positive and negative control with inducible and repressible . Copyright © 2013 by Jones & Bartiett Learning, LLC an Ascend Learning Company COntrOI. www.jblearning.com Spare slides! Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com DNA is superior to dsRNA as a genomic material because of the major groove. Specific base recognition by proteins is easy in DNA. dsRNA A-helix Deep and narrow Major grrove. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Wide major groove means DNA sequence can be recognized by DNA-binding proteins. major groove major groove FIGURE 6-10 Chemical groups exposed in the major and minor grooves from the edges of the base pairs. The letters in red identify hydrogen bond acceptors (A), hydrogen bond donors (D), nonpolar hydrogens (H), and methyl groups (M). minor groove minor groove ■any :om 26.1 Introduction • regulator gene - A gene that codes for a product (typically protein) that controls the expression of other genes (usually at the level of transcription). • structural gene - A gene that codes for any RNA or protein product other than a regulator. Figure 26.01: A regulator gene codes for a protein that acts at a target site on DNA. \/\/\/ mRNA Regulator protein Target site Structural gene Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.1 Introduction trans-acting - A product that can function on any copy of its target DNA. This implies that it is a diffusible protein or RNA. c/s-acting - A site that affects the activity only of sequences on its own molecule of DNA (or RNA); this property usually implies that the site does not code for protein. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com 26.1 Introduction 3 a V) rr NEGATIVE CONTROL sm^r> s\/\ I Repressor REPRESSED I Inac Inactive 9 repressor Inducer INDUCED Inactive repressor Corepressor Active repressor INDUCED T-PRESSED POSITIVE CONTROL RNA polymerase 4 J r Inactive | Active activator Q activator Inducer REPRESSED INDUCED - Active activator r INDUCED Inactive • activator Corepressor REPRESSED Figure 26.04: Regulatory circuits can be designed from all possible combinations of positive and negative control with inducible and repressible control. Gene regulation in vivo can utilize any of these mechanisms, resulting in all four combinations: negative inducible, negative repressible, positive inducible, and positive repressible. Copyright © 2013 by Jones & Bartiett Learning, LLC an Ascend Learning Company www.jblearning.com Beginnings of genetics in Saccharomyces cerevisiae. • Yeast genetics began only in the 1950s - much later than E. coli genetics. Commercial strains of brewer's and baker's yeast are stable diploids or tetraploids and could not be studied genetically. • Occasional meiosis in diploid stains gives an ascus containing four haploid spores two with a and two with a mating types but these immediately mate back to diploids (Ascomycete family of fungi). Pure haploid cultures could not be grown from spores because after the first haploid cell division the mother cell switches mating type and can mate with cell it just budded off. • Yeast genetics required mutants in a gene called HO (homothallism - is a plant term for having both types of sexual parts on one organism). The ho mutation keep haploid cell lines stable because it prevents mating type switching. Winge, (Carlsberg labs, Copenhagen), Lindegren, (Anheuser Busch, Carbondale, Illinois). • Much of yeast genetics is done with the haploid strains that have only one copy of each gene (same as E. coli) but can be mated to give diploids and sporulated back to haploids by nitrogen starvation. Copyright © 2013 by Jones & Bartlett Learning, LLC an Ascend Learning Company www.jblearning.com Genome sizes increase faster than gene numbers among the main model organisms. Sequenced genomes vary from 470 to 30,000 genes Species Genomes (Mb) Genes Lethal loci Mycoplasma genitalium 0.58 470 -300 Rickettsia prowazekii 1.11 S34 Haemophilus influenzae 1.83 1r743 Methanococcus jannascfti 1.66 1,733 B. subtiiis 4.2 4,100 E. coli 4.6 4,288 1,800 S. cerevisiae 13.5 6,034 1,090 S. pombe 12.5 4,929 A. thaliana 119 25,498 O. sativa (rice) 466 -30,000 D. melanogaster 165 13,601 3,100 C. elegans 97 18r424 H. sapiens 3,300 -25,000 Bacterial minimum 1800. Yeast has 6,000 genes. About 2X basic eukaryotic minimum set of 2,700 or so. Simpler animals like Drosophila have 2X as many genes as yeast. Humans and other mammals have only 2X Drosophila. 4,600 kb 13,500 kb 3x genome size of E. coli 165,000 kb 10X genome size of yeast 3,000,000 kb 20X Drosophila genome fright ©20i" - ■ a 3 by Jones & Barttett Learning, LLC an Ascend Learning Company www.jblearning.com