Prokaryotic Gene Regulation PDF

Concepts of Genetics Twelfth Edition Chapter 16 Regulation of Gene Expression in Bacteria Copyright © 2021, 2019, 2015 Pearson Educa...

Concepts of Genetics Twelfth Edition Chapter 16 Regulation of Gene Expression in Bacteria Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved 16.1 Bacteria Regulate Gene Expression in Response to Environmental Conditions Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Prokaryote Gene Regulation Let’s think about differences between prokaryotic cells and eukaryotic cells (including differences in the size and organization of their genomes). How might the processes used by these organisms to regulate gene expression reflect these differences? Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Prokaryote Gene Regulation E. coli gene regulation has been studied extensively – Highly efficient genetic mechanisms have evolved that turn transcription of specific genes on and off depending on metabolic need for gene products – Respond to changes in environment – Regulate gene activity—replication, recombination, DNA repair, and cell division Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Inducible and Constitutive Enzymes Constitutive enzymes – Enzymes are continuously produced regardless of chemical makeup of environment Inducible enzymes – Bacteria adjust to environment by producing inducible enzymes only when specific substrates are present (only when needed) Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Repressible System Repressible system – Presence of a specific molecule inhibits gene expression – For example, abundance of end product of a process may repress expression of genes involved in the making of that product. Thus, if enough of the product is present, it can repress the expression of genes needed to make that product until levels of the product go down again Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Positive and Negative Control Regulation of inducible or repressible type system under positive control or negative control – Positive control: Transcription occurs only when regulator molecule directly stimulates RNA production (transcription) – Negative control: Genetic expression occurs unless shut off by regulator molecule Either type of system, or both combined, can induce or repress systems Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved 16.2 Lactose Metabolism in E. Coli is Regulated by an Inducible System Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Lactose Metabolism Lactose: Galactose and glucose containing disaccharide – In prokaryotes, gene activity is repressed when lactose is absent and induced when available – In the presence of lactose, the concentration of enzymes responsible for its metabolism increases = inducible enzymes Lactose is the inducer that promotes expression of genes needed to take lactose up into the cell and break it down. Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Regulatory Regions Transcription is under control of single regulatory region – In prokaryotes, genes coding for enzymes with regulatory functions are organized in clusters – Regulatory regions usually located upstream of gene cluster they control – Regulatory region on same strand—cis-acting – Trans-acting elements: Molecules that bind cis- acting sites Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Cis- and Trans-Acting Sites Cis- and trans-acting sites – Regulatory site events determine if genes are transcribed into mRNA – Binding of trans-acting element (such as a protein) at cis-acting site (usually a DNA sequence) regulates genes cluster negatively or positively ▪ Negatively by turning off transcription ▪ Positively by turning on transcription Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Operons Operons are a collection of genes controlled by a regulatory region. Operons consist of four basic parts: – Promoter—genomic site recognized and bound by RNA polymerase to start transcription. – Genes—operons have 2 or more genes that work together in a metabolic pathway – Repressor—a protein produced from a different genomic location that blocks transcription – Operator—a regulatory region upstream of genes in operon, binds repressor to block transcription. https://journals.asm.org/doi/10.1128/jmbe.00006-23 (Also source for class activity) Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Lac Operon (1 of 2) Lac (lactose) operon – Has three structural genes: lacZ, lacY, and lacA – Operon has upstream regulatory region consisting of operator and promoter Entire gene cluster functions to provide rapid response to presence or absence of lactose The default expression is “off”. When in the presence of lactose, the operon is turned “on.” Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved  Lac Operon Lac (lactose) operon – Has three structural genes: lacZ, lacY, and lacA – Operon has upstream regulatory region consisting of operator and promoter Entire gene cluster functions to provide rapid response to presence or absence of lactose Note that the Z, Y, and A genes will be transcribed into a single mRNA, a condition referred to as polycistronic. Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved lacZ and  -Galactosidase (1 of 2) beta lacZ gene – Encodes beta-galactosidase, an enzyme that converts disaccharide lactose to monosaccharides glucose and galactose – Conversion is necessary for lactose to serve as primary energy source in glycolysis Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Section 16.2 lacY and lacA lacY gene - Codes for permease, an enzyme that facilitates entry of lactose into bacterial cell lacA – Encodes enzyme transacetylase, which may be involved in removal of toxic by-products of lactose digestion from the cell Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved  Lac operon structural genes—lacZ, lacY, and lacA – All three are transcribed as single unit – Results in polycistronic m RNA – Cistron: Part of nucleotide sequence coding for single gene – Single mRNA is translated into three gene products Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved lacI Repressor Gene lacI gene – Located close to but not part of lac operon structural genes – Produces repressor molecule, which regulates transcription of structural genes – Allosteric repressor is normally produced by lacl ▪ Interacts reversibly with operator region ▪ Causes conformational change in three- dimensional shape and change in chemical activity Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Section 16.2 Operon Model: Negative Control Jacob and Monod (1960) – Proposed operon model: ▪ Group of genes is regulated and expressed together as a unit – Proposed lacI gene regulates transcription of structural genes by producing allosteric repressor molecule Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Section 16.2 Negative Control (1 of 2) Lac operon: Negative control – Operon subject to negative control: Transcription occurs only when repressor fails to bind operator region – Repressor normally binds DNA sequence in operator region ▪ Inhibits RNA polymerase ▪ Represses transcription of structural genes Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Negative Control Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Summary of Operon Model (1 of 2) Operon model – Invokes series of molecular interactions between proteins, inducers, and DNA – No lactose: Enzymes are not needed and expression of genes encoding enzymes is repressed – Lactose present: Indirectly induces activation of genes by binding repressor – All lactose metabolized: None is available to bind to repressor and transcription is repressed Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Genetic Proof of Operon Model Major predictions of operon model – I gene produces diffusible product (trans-acting protein) – O (operator) region is a DNA sequence (cis-acting) involved in regulation – O region must be adjacent to structural genes to regulate transcription Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved  Constitutive I super minus and O su mutations in I- and OC interfere Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Be familiar with the consequences of each of the following mutations Both the I  and OC constitutive mutations interfere with the molecular interactions – The structural genes are always turned on I  mutant: The repressor protein is altered or absent and cannot bind to the operator region – The structural genes are always turned on OC mutant: The nucleotide sequence of the operator DNA is altered and will not bind with a normal repressor molecule – The structural genes are always turned on Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved  TABLE 16.1 A Comparison of Gene Activity  or   in the Presence or Absence of Lactose for Various E. coli Genotypes Presence of  -Galactosidase Activity Genotype Lactose Present Lactose Absent A. I +O + Z + I super plus O super plus Z super plus + negative I +O + Z  I super plus O super plus Z super minus  negative  negative I  O+Z + I super minus O super plus Z super plus + + I +OC Z + I super plus O super C Z super plus + + B. I  O  Z  / F′I  I super minus O super plus Z super plus over F prime I super plus +  negative I OC Z  / F′O  I super plus O super C Z super plus over F prime O super plus + + C. I O  Z  / F′I  I super plus O super plus Z super plus over F prime I super minus +  negative I O  Z  / F′OC I super plus O super plus Z super plus over F prime O super C +  negative D. I SO  Z  I super S O super plus Z super plus  negative  I SO  Z  / F ′I  I super S O super plus Z super plus over F prime I super plus  negative  negative *Note: In parts B to D, most genotypes are partially diploid, containing an F factor plus attached genes F′  Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved 16.3 The Catabolite-Activating Protein (CAP) Exerts Positive Control over the lac Operon Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Section 16.3 CAP (1 of 2) CAP: Catabolite-activating protein can also regulate the operon – helps detect and respond to glucose, the preferred energy source for the bacterial – Exerts positive control over lac operon – Diminishes expression of operon when glucose is present (catabolite repression) – The cell prefers to use glucose. If it is present, there is no need to break down lactose – If glucose is low, binds to CAP-binding site, facilitating RNA polymerase binding at promoter and facilitating transcription Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Section 16.3 Transcription Levels Maximum transcription of structural genes requires 2 things: – Repressor must be bound by lactose ▪ Does not repress operon expression – CAP must be bound to CAP-binding site to most efficiently promote expression of the operon by promoting association of RNA polymerase with the promoter Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Section 16.3 Glucose Inhibits CAP Binding (1 of 3) cAMP: Cyclic adenosine monophosphate – To bind to promoter, CAP must be bound to cyclic adenosine monophosphate (cAMP) – Glucose inhibits activity of adenylyl cyclase, which catalyzes conversion of ATP to cAMP – Prevents CAP from binding when glucose is present – In this way, cAMP acts as a molecular sensor to reflect levels of glucose (high glucose = low cAMP, low glucose=high cAMP) Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Glucose Inhibits CAP Binding Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved 16.5 The Tryptophan (tr p) Operon in E. coli is a Repressible Gene System Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Section 16.5 Tryptophan (trp) Operon Tryptophan (trp) operon – Repressible gene system in E. coli – Wild-type E. coli produce enzymes for biosynthesis of amino acids and essential macromolecules – Tryptophan present: Enzymes necessary for synthesis of tryptophan are not produced – No need for the organism to waste energy and other resources to produce enzymes when they are not needed Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Section 16.5 Structural Genes of trp Operon (1 of 2) Structural genes of trp operon Five contiguous structural genes transcribed as polycistronic message – Involved in tryptophan production ▪ trpE, D, C, B, A – Enzymes catalyze biosynthesis of tryptophan trpP: promoter—binding site for RNA Pol trpO: operator sequence—binds repressor protein. Repressor can only bind when it is bound to tryptophan (indicates that tryptophan is present, and the operon will not be transcribed as no additional tryptophan is needed Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved 16.6 R N A Plays Diverse Roles in Regulating Gene Expression in Bacteria Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved  Riboswitches can also regulate gene expression in bacteria – Alternative forms of mRNA secondary structure – Bind with small ligands; cause conformational change and induce second RNA domain – Create antiterminator or terminator structure Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved  Bacterial small noncoding RNAs can regulate gene expression – Can be negative regulators of gene expression by binding to mRNAs and preventing translation by masking the ribosome- binding site (RBS) Copyright © 2021, 2019, 2015 Pearson Education, Inc. All Rights Reserved  Bacterial small noncoding RNAs can regulate gene expression – Can be positive regulators of gene expression by binding to mRNAs and preventing secondary structures (that would otherwise mask an RBS) and enable translation Copyright © 2021, 2019, 2015 Pearson Education, Inc. 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