Gene Expression Regulation In Prokaryotes PDF

1  2 PART II – E.1 Control of gene expression (Prokaryotes)  3 In bacteria, regulation of gene expression occurs at several levels: 1. Genome/chromatin structure 2. Epigenetic profile 3. Transcriptional regulation 4...

1  2 PART II – E.1 Control of gene expression (Prokaryotes)  3 In bacteria, regulation of gene expression occurs at several levels: 1. Genome/chromatin structure 2. Epigenetic profile 3. Transcriptional regulation 4. Post-transcriptional regulation 5. Regulation of Translation 6. Regulation via non-coding RNAs  4 Chromatin Structure Nucleoid shape changes in different situations by NAPs and lncRNAs. NAPs have both structural and regulatory functions: Modify chromosome architecture Mediate interactions with ncRNAs Moderate transcription: by blocking promoter, advancement of RNAP, positive supercoiling, binding of factors to nascent RNA Xenogenic silencing Regulation of virulence Transcription also impacts nucleoid structure: Template needs to be available to RNAP Generation of positive supercoils in dsDNA 5  6 Epigenetic profile – DNA methylation Adenine methylation Restriction modification (R-M system) Endogenous methylases Protects bacterial chromosome against its own endonucleases Cleaves phage DNA Orphan methylases (GATC sites) can regulate: Gene expression: methylation allows binding of activators or repressors. DNA repair: methyl-directed MMR machinery can recognize the template strand. Initiation of DNA replication: hemimethylation at promoter of dnaA and oriC allows binding of a replication initiation regulator protein (SeqA) to prevent unwanted reinitiation.  7 Endonuclease Methylase 8  9 Transcriptional regulation Bacterial genes are often organized into operons: Proteins of same functional pathway are regulated and synthesized together. Bacteria can respond quickly to changes in the environment. Depending on how operons respond to specific compounds the environment: Inducible operons: transcription happens in the presence of the substrate. Repressible operons: transcription is repressed in the presence of the product. Modes of transcriptional regulation and examples will be described later  10 + Substrate = RNAP binds promoter Effect of Inducible S turns transcription ON Type of operon (catabolic) substrate (System) or product Repressible P turns transcription OFF (anabolic) + Product = RNAP cannot bind promoter  11 Post-transcriptional regulation RNA editing: rNTPs are modified after transcription Changes properties of the rNTP Can alter amino acid sequence Ex: Adenosine to Inosine = ribosome recognizes I as G (changes the codon) RNA turnover: balance between synthesis and degradation. RNA quantity, availability and turnover can be modified by: Abundance and location of RNases Binding to ncRNAs and other molecules 12 13  14 Regulation of Translation Many mechanisms of translational regulation overlap with transcriptional regulation Presence and strength of ribosome binding sites on the mRNA Codon usage bias Post-translational editing  15 Genomic data set of 327 species that covers nearly one third of the known biodiversity of the budding yeast subphylum Saccharomycotina  16 Regulation via non-coding RNAs Most studied mechanisms involve small ncRNAs Gene expression: Transcription and translation mRNA stability Stress response Virulence Xenogenic silencing  17 Examples of regulation via sRNAs Cis-encoded ncRNA elements: Structural motifs found in the 5’ UTR that responds to cellular or environmental changes. Examples: attenuation, leader peptides, thermosensors, riboswitches. Cis-encoded antisense RNAs: Target the mRNA for degradation. Encoded in or close to the gene but produced from an antisense promoter. Trans-encoded antisense ncRNAs: Target the mRNA to impact transcription and translation. Encoded in a different genomic locus from the gene they regulate.  18 Riboswitches  19 Cis or trans encoded antisense RNAs  20 PART II – E.2 Control of gene expression (Prokaryotes)  21 Modes of transcriptional regulation Availability of sigma Binds UP element factor and RNAP Binds close to promoter to facilitate sigma factor binding Activators (positive control) DNA conformation change to facilitate sigma factor binding Regulators Prevents repressor binding or (transcription factors) repressor function Regulation Steric hindrance Repressors Road block (positive control) DNA looping Modulation of activator Attenuation Pausing or terminating transcription Antitermination RNAP forgoes initial termination signal 22 23 24  25 Transcriptional Regulation Effect of Inducible S turns transcription ON Type of operon (catabolic) substrate (System) or product Repressible P turns transcription OFF (anabolic) Binds promoter Positive Transcription ON Aids RNA Pol Function of Control Regulator Binds operator Negative Transcription OFF Blocks RNA Pol Regulators can be active or inactive immediately after synthesis Regulators can become active (can bind DNA) or inactive (cannot bind DNA) by binding a cofactor 26  27 INDUCIBLE OPERON UNDER POSITIVE CONTROL Positive Inducible control Substrate and Activator must work TOGETHER Activator If SUBSTRATE is to activate transcription present, transcription is ON Aids binding of RNA Pol when attached to promoter = transcription is ON CONCLUSION: When the substrate is available in the environment, it activates the activator, which binds the promoter, aiding the attachment of the RNA Pol to the promoter. This leads to transcription of the structural genes of the operon.  28 REPRESSIBLE OPERON UNDER POSITIVE CONTROL Positive Repressible control Product and Activator have opposite effects Activator on transcription If PRODUCT is present, transcription is OFF Aids binding of RNA Pol when attached to promoter = transcription is ON CONCLUSION: When the product is available in the environment, it inactivates the activator so it cannot bind the promoter. In these conditions, RNA Pol cannot bind the promoter either, hence structural genes of the operon are not transcribed.  29 lac OPERON 30  31 Lactose absent  32 Lactose present (no glucose)  33 Carbon catabolite repression (positive control) r= ein g u lato tor prot = re ctiva CAP olite a b cata  34 trp OPERON  35 If tryptophan concentration is high Produces an inactive repressor No transcription W Active W W repressor W W = tryptophan Attenuation is a second mechanism of negative feedback in the trp operon  36 141 bp Stalls RNAP. Ribosome initiates translation of leader peptide, pushing RNAP. RNAP and ribosome couple.  37 ribosome in region 2 low trp high trp ribosome is slow ribosome is fast 38  39 Low W concentration 2:3: If W concentration is LOW: Ribosome moves slowly through leader peptide. Transcription and translation become uncoupled. Antiterminator hairpin forms: structural genes are transcribed.  40 High W concentration 3:4: If W concentration is HIGH: Ribosome moves quickly through leader and stays coupled with RNAP. Since ribosome is on region 2, regions 2 and 3 cannot interact. Hairpin 3:4 is an attenuator and terminates transcription.  41 Mutation studies and merozygotes (partial diploids) E. coli cell with chromosome e f g Plasmid f g  42 ADDITIONAL INFORMATION & EXTRA SLIDES  43 Arginine operon In minimum media, E. coli normally produces the enzymes required for synthesis of arginine. If arginine is provided in the media, E. coli stops producing the enzymes. Mutations in the regulator gene lead to continuous transcription of the structural genes of the operon even when arginine is provided. Is this an inducible or repressible operon? Is it under positive or negative control?  44 Maltose operon Enzymes for maltose catabolism in E. coli are synthesized only after the addition of maltose to the medium. Mutations in the regulator gene eliminate production of enzymes required for maltose catabolism, both in the presence and absence of maltose. Is this an inducible or repressible operon? Is it under positive or negative control?  45 Application Exercise For E. coli strains with the following lac genotypes, use a plus sign (+) to indicate the synthesis of β-galactosidase, permease, and transacetylase and a minus sign (-) to indicate no synthesis of these enzymes. Genotype of strain Lactose present Lactose absent β-gal Perm Trans β-gal Perm Trans lacI+ lacP+ lacO+ lacZ+ lacY+ lacI+ lacP+ lacOc lacZ-- lacY+ lacI+ lacP-- lacO+ lacZ+ lacY-- lacI+ lacP+ lacO+ lacZ-- lacY— /lacI-- lacP+ lacO+ lacZ+ lacY+  46 Levels of Gene Regulation REGULATION PROKARYOTES EUKARYOTES Chromatin level Transcription level mRNA processing level Regulation of RNA stability (amount synthesized, degradation rate) Level of translation (presence of factors, amino acids) Post-translational modifications  47 INDUCIBLE OPERON UNDER POSITIVE CONTROL Positive control Inducible Substrate and Activator Activator must work TOGETHER to activate transcription If substrate is present, transcription is ON Aids binding of RNA Pol when attached to promoter = transcription is ON CONCLUSION: When the substrate is available in the environment, it activates the activator, which in turn binds the promoter to aids attachment of the RNA Pol to the promoter. This leads to transcription of the structural genes of the operon.  48 REPRESSIBLE OPERON UNDER NEGATIVE CONTROL Negative control Repressible Product and Repressor Repressor must work TOGETHER to inactivate transcription If product is present, transcription is OFF Prevents binding of RNA Pol when attached to operator = transcription is OFF CONCLUSION: When the product is available in the environment, it activates the repressor, which in turn binds the operator to prevent attachment of the RNA Pol to the promoter. Structural genes of the operon are not transcribed.  49 INDUCIBLE OPERON UNDER NEGATIVE CONTROL Negative control Inducible Substrate and Repressor Repressor have opposite effects on transcription If substrate is present, transcription is ON Prevents binding of RNA Pol when attached to operator = transcription is OFF CONCLUSION: When the substrate is available in the environment, it inactivates the repressor. In these conditions, RNA Pol can bind the promoter, which leads to transcription of the structural genes of the operon.  50 REPRESSIBLE OPERON UNDER POSITIVE CONTROL Positive control Repressible Product and Activator Activator have opposite effects on transcription If product is present, transcription is OFF Aids binding of RNA Pol when attached to promoter = transcription is ON CONCLUSION: When the product is available in the environment, it inactivates the activator so it cannot bind the promoter. In these conditions, RNA Pol cannot bind the promoter either, hence structural genes of the operon are not transcribed. 51 52  53 Clicker questions:  54 For any organism, regulation of gene expression can occur at several stages, including: a. transcription b. translation c. posttranslational modification d. mRNA processing and stability e. all of the above  55 A ________ gene has a product that interacts with other nucleotide sequences, affecting gene expression a. constitutive b. regulatory c. structural d. none of the above  56 A typical operon contains several regions. Which region is where a regulator protein binds? a. structural genes b. promoter c. operator d. none of the above  57 In an inducible operon, the presence of the substrate activates transcription: a. true b. false  58 In a repressible operon, the presence of the product in the environment turns transcription off: a. true b. false  59 Inducible or Repressible Operons = Effect of substrate/product on state of transcription  60 In an operon under positive control, when active, the activator aids the binding of RNA Pol to the promoter: a. true b. false  61 In an operon under negative control, when active, the activator aids the binding of RNA Pol to the promoter: a. true b. false  62 Negative or Positive Control = effect of regulator on RNAP’s attachment to promoter  63 In a negative repressible operon, the product will bind to the repressor and activate it. This turns ‘off’ transcription of the structural genes: a. true b. false  64 In a positive inducible operon, transcription of the structural genes is turned off in presence of the substrate a. true b. false  65 In a negative inducible operon, the regulatory molecule is a repressor that, in absence of the substrate, does not let RNA polymerase interact with the promoter: a. true b. false  66 In a positive repressible operon, the regulatory protein acts as an activator, aiding the interaction of RNA polymerase with the promoter when there is no product: a. true b. false  67 To activate the transcription of permease (and the whole lac operon) lactose needs to be present in the cell. How can lactose get in the cell when the permease gene is not activated? a. The repressed lac operon is never completely repressed. b. There is a second permease in the genome that initially transports lactose. c. Lactose freely moves across the plasma membrane. d. Though the lac operon has been studied for many years, this mystery is unsolved.  68 Which element of the lac operon acts in cis? a. Operator b. Regulatory molecule c. Betagalactosidase, permease, transacetylase d. Promoter  69 Which factor of the lac operon acts in trans? a. Operator b. Regulatory molecule c. Betagalactosidase, permease, transacetylase d. Promoter  70 An example of a gene product encoded by a regulatory gene is a. beta-galactosidase enzyme. b. allolactose. c. repressor protein. d. operator.  71 Where would the lac repressor be bound in a (nonmutant) E. coli cell that is growing in no glucose and high lactose? a. P b. O c. P and O d. I, P, O e. The repressor would not be bound.  72 A mutant E. coli strain, grown under conditions that normally induce the lac operon, produces high amounts of ß-galactosidase. What is a possible genotype of the cells? a. lacI+ lacP+ lacO+ lacZ– lacY+ lacA+ b. lacI+ lacP+ lacOc lacZ+ lacY+ lacA+ c. lacI– lacP+ lacO+ lacZ– lacY+ lacA+ d. lacI+ lacP– lacO+ lacZ+ lacY+ lacA+  73 A mutant E. coli strain, grown under conditions that normally induce the lac operon, does not produce ß-galactosidase. What is a possible genotype of the cells? a. lacI+ lacP+ lacO+ lacZ+ lacY– lacA+ b. lacI+ lacP+ lacOc lacZ+ lacY+ lacA+ c. lacl+ lacP+ lacO+ lacZ+ lacY+ lacA+ d. lacI+ lacP– lacO+ lacZ+ lacY+ lacA+  74 Other questions (trp operon) What would be the consequence of deleting region 1 from the 5’ UTR of the trp operon? Same question can be asked for regions 2, 3, 4, or mutations in trpR.  75 WORKSHEET - Conclusions Presence of Regulator is an Type of operon (substrate/product) turns (activator/repressor) transcription (on/off) Negative inducible SUBSTRATE - ON REPRESSOR Positive inducible SUBSTRATE - ON ACTIVATOR Negative repressible PRODUCT - OFF REPRESSOR Positive repressible PRODUCT - OFF ACTIVATOR 76 77 78