Control of Gene Expression in Prokaryotes Benjamin A. Pierce GENETICS A Conceptual Approach FIFTH EDITION CHAPTER 16 Control of Gene Expression in Prokaryotes © 2014 W. H. Freeman and Company 1

The expression of genes in bacteria is often regulated through operons, groups of genes that are transcribed as a unit. Shown here is Escherichia coli, a common bacterium found in the intestinal tracts of mammals.

16.1 The Regulation of Gene Expression is Critical for All Organisms Genes and Regulatory Elements Levels of Gene Regulation DNA-Binding Proteins

Genes and Regulatory Elements Structural genes: encoding proteins Regulatory genes: encoding products that interact with other sequences and affect the transcription and translation of these sequences Regulatory elements: DNA sequences that are not transcribed but play a role in regulating other nucleotide sequences

Genes and Regulatory Elements Constitutive expression: continuously expressed under normal cellular conditions Positive control: stimulate gene expression Negative control: inhibit gene expression

Levels of Gene Regulation Figure 16.1 Gene expression can be controlled at multiple levels.

DNA-Binding Proteins Domains: ~ 60- 90 amino acids, responsible for binding to DNA, forming hydrogen bonds with DNA Motif: within the binding domain, a simple structure that fits into the major groove of the DNA Distinctive types of DNA-binding proteins based on the motif

Figure 16.2 DNA-binding proteins can be grouped into several types on the basis of their structures, or motifs.

Concept Check 1 How do amino acids in DNA-binding proteins interact with DNA? By forming covalent bonds with DNA base By forming hydrogen bonds with DNA base By forming covalent bonds with sugars

Concept Check 1 How do amino acids in DNA-binding proteins interact with DNA? By forming covalent bonds with DNA base By forming hydrogen bonds with DNA base By forming covalent bonds with sugars

16.2 Operons Control Transcription in Bacterial Cells Operon Structure Negative and Positive Control: Inducible and Repressible Operons The lac Operon of E. coli lac Mutations Positive Control and Catabolite Repression The trp Operon of E. coli

Operon Structure Operon: promoter + additional sequences that control transcription (operator) + structure genes Regulator gene: DNA sequence encoding products that affect the operon function, but are not part of the operon

Figure 16.3 An operon is a single transcriptional unit that includes a series of structural genes, a promoter, and an operator.

Concept Check 2 What is the difference between a structural gene and a regulator gene? Structural genes are transcribed into mRNA, but regulator genes are not. Structural genes have complex structures; regulator genes have simple structure. Structural genes encode proteins that function in the structure of the cell; regulator genes carry out metabolic reactions. Structural genes encode proteins; regulator genes control the transcription of structural genes.

Concept Check 2 What is the difference between a structural gene and a regulator gene? Structural genes are transcribed into mRNA, but regulator genes are not. Structural genes have complex structures; regulator genes have simple structure. Structural genes encode proteins that function in the structure of the cell; regulator genes carry out metabolic reactions. Structural genes encode proteins; regulator genes control the transcription of structural genes.

Negative and Positive Control: Inducible and Repressible Operons Inducible operons: Transcription is usually off and needs to be turned on. Repressible operons: Transcription is normally on and needs to be turned off.

Negative and Positive Control: Inducible and Repressible Operons Negative inducible operons: The control at the operator site is negative. Molecule binding is to the operator, inhibiting transcription. Such operons are usually off and need to be turned on, so the transcription is inducible. Inducer: small molecule that turns on the transcription.

Figure 16.4 Some operons are inducible.

Negative and Positive Control: Inducible and Repressible Operons Negative repressible operons: The control at the operator site is negative. But such transcription is usually on and needs to be turned off, so the transcription is repressible. Corepressor: a small molecule that binds to the repressor and makes it capable of binding to the operator to turn off transcription.

Figure 16.5 Some operons are repressible.

Negative and Positive Control: Inducible and Repressible Operons Positive inducible operon Positive repressible operon

Figure 16.6 A summary of the characteristics of positive and negative control in inducible and repressible operons.

Concept Check 3 In a negative repressible operon, the regulator protein is synthesized as . an active activator an inactive activator an active repressor an inactive repressor

Concept Check 3 In a negative repressible operon, the regulator protein is synthesized as . an active activator an inactive activator an active repressor an inactive repressor

A negative inducible operon Lactose metabolism The lac Operon of E. coli A negative inducible operon Lactose metabolism Regulation of the lac operon Inducer: allolactose lacI: repressor encoding gene lacP: operon promoter lacO: operon operator

The lac Operon of E. coli Structural genes lacZ: encoding β-galactosidases lacY: encoding permease lacA: encoding transacetylase The repression of the lac operon never completely shuts down transcription.

Figure 16.7 Lactose, a major carbohydrate found in milk, consists of two six-carbon sugars linked together

Figure 16.8 The lac operon regulates lactose metabolism.

Figure 16.9 In the lac operon, the operator overlaps the promoter and the 5′ end of the first structural gene.

In the presence of allolactose, the lac repressor . Concept Check 4 In the presence of allolactose, the lac repressor . binds to the operator binds to the promoter cannot bind to the operator binds to the regulator gene

In the presence of allolactose, the lac repressor . Concept Check 4 In the presence of allolactose, the lac repressor . binds to the operator binds to the promoter cannot bind to the operator binds to the regulator gene

lac Mutations Partial diploid: full bacterial chromosome + an extra piece of DNA on F plasmid Structural gene mutations: affect the structure of the enzymes, but not the regulations of their synthesis lacZ+lacY−/lacZ−lacY+ produce fully functional β-galactosidase and permease.

lac Mutations Regulator gene mutations: lacI− leads to constitutive transcription of three structure genes. lacI+ is dominant over lacI− and is trans acting. A single copy of lacI+ brings about normal regulation of lac operon. lacI+lacZ−/lacI−lacZ+ produce fully functional β-galactosidase.

Figure 16.10 The partial diploid lacI + lacZ −/lacI − lacZ + produces β-galactosidase only in the presence of lactose because the lacI gene is trans dominant.

Figure 16.11 The partial diploid lacI s lacZ +/lacI + lacZ + fails to produce β-galactosidase in the presence and absence of lactose because the lacIs gene encodes a superrepressor.

lac Mutations Operator mutations: lacOc: C = constitutive lacOc is dominant over lacO+, which is cis acting. lacI+lacO+Z−/lacI+lacOclacZ+ produce fully functional β-galactosidase constitutively.

Figure 16. 12 Mutations in lacO are constitutive and cis acting Figure 16.12 Mutations in lacO are constitutive and cis acting. (a) The partial diploid lacI + lacO + lacZ −/ lacI + lacOc lacZ+ is constitutive, producing β-galactosidase in the presence and absence of lactose. (b) The partial diploid lacI + lacO + lacZ+/lacI + lacOc lacZ− is inducible (produces β-galactosidase only when lactose is present), demonstrating that the lacO gene is cis acting.

lac Mutations Promoter mutations lacP−: cis acting lacI+lacP−lacZ+/lacI+lacP+lacZ− fails to produce functional β-galactosidase.

Characteristics of lac operon mutations.

Positive Control and Catabolite Repression Catabolite repression: using glucose when available, and repressing the metabolite of other sugars. This is a positive control mechanism. The positive effect is activated by catabolite activator protein (CAP). cAMP is binded to CAP, together CAP–cAMP complex binds to a site slightly upstream from the lac gene promoter.

Positive Control and Catabolite Repression cAMP: adenosine-3′, 5′-cyclic monophosphate The concentration of cAMP is inversely proportional to the level of available glucose.

Figure 16.13 The catabolite activator protein (CAP) binds to the promoter of the lac operon and stimulates transcription. CAP must complex with adenosine-3′, 5′-cyclic monophosphate (cAMP) before binding to the promoter of the lac operon. The binding of cAMP–CAP to the promoter activates transcription by facilitating the binding of RNA polymerase. Levels of cAMP are inversely related to glucose: low glucose stimulates high cAMP; high glucose stimulates low cAMP.

Figure 16.14 The binding of the cAMP–CAP complex to DNA produces a sharp bend in DNA that activates transcription.

What is the effect of high levels of glucose on the lac operon? Concept Check 5 What is the effect of high levels of glucose on the lac operon? Transcription is stimulated. Little transcription takes place. Transcription is not affected. Transcription may be stimulated or inhibited, depending on the levels of lactose.

What is the effect of high levels of glucose on the lac operon? Concept Check 5 What is the effect of high levels of glucose on the lac operon? Transcription is stimulated. Little transcription takes place. Transcription is not affected. Transcription may be stimulated or inhibited, depending on the levels of lactose.

The trp Operon of E. coli A negative repressible operon Five structural genes trpE, trpD, trpC, trpB, and trpA—five enzymes together convert chorismate to typtophane.

Figure 16.15 The trp operon controls the biosynthesis of the amino acid tryptophan in E. coli.

Concept Check 6 In the trp operon, what happens to the trp repressor in the absence of tryptophan? It binds to the operator and represses transcription. It cannot bind to the operator and transcription takes place. It binds to the regulator gene and represses transcription. It cannot bind to the regulator gene and transcription takes place.

Concept Check 6 In the trp operon, what happens to the trp repressor in the absence of tryptophan? It binds to the operator and represses transcription. It cannot bind to the operator and transcription takes place. It binds to the regulator gene and represses transcription. It cannot bind to the regulator gene and transcription takes place.

16.3 Some Operons Regulate Transcription Through Attenuation, the Premature Termination of Transcription Attenuation: affects the continuation of transcription, not its initiation. This action terminates the transcription before it reaches the structural genes. Attenuator Antiterminator

Attenuation in the trp Operon of E. coli Four regions of the long 5′ UTR (leader) region of trpE mRNA When tryptophan is high, region 1 binds to region 2, which leads to the binding of region 3 and region 4, terminating transcription prematurely.

Figure 16.16 Two different secondary structures can be formed by the 5′ UTR of the mRNA transcript of the trp operon.

Attenuation in the trp Operon of E. coli Four regions of the long 5′ UTR (leader) region of trpE mRNA When tryptophan is low, region 2 binds to region 3, which prevents the binding of region 3 and region 4, and transcription continues.

Figure 16.17 Whether the premature termination of transcription (attenuation) takes place in the trp operon depends on the cellular level of tryptophan.

Events in the process of attenuation.

16.4 RNA Molecules Control the Expression of Some Bacterial Genes Antisense RNA: complementary to targeted partial sequence of mRNA Riboswitches: molecules influence the formation of secondary structures in mRNA Ribozymes: mRNA molecules with catalytic activity

Figure 16.18 Antisense RNA can regulate translation.

Figure 16.19 Riboswitches are RNA sequences in mRNA that affect gene expression.