PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker CHAPTER 14 GENE REGULATION IN BACTERIA AND BACTERIOPHAGES Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

INTRODUCTION The term gene regulation means that the level of gene expression can vary under different conditions Genes that are unregulated are termed constitutive They have essentially constant levels of expression Frequently, constitutive genes encode proteins that are necessary for the survival of the organism The benefit of regulating genes is that encoded proteins will be produced only when required 14-2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

INTRODUCTION Gene regulation is important for cellular processes such as 1. Metabolism 2. Response to environmental stress 3. Cell division Regulation can occur at any of the points on the pathway to gene expression Refer to Figure 14.1 14-3 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 14.1 14-4

14.1 TRANSCRIPTIONAL REGULATION The most common way to regulate gene expression in bacteria is at the transcriptional level The rate of RNA synthesis can be increased or decreased Transcriptional regulation involves the actions of two main types of regulatory proteins Repressors  Bind to DNA and inhibit transcription Activators  Bind to DNA and increase transcription Negative control refers to transcriptional regulation by repressor proteins Positive control to regulation by activator proteins 14-5 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Small effector molecules affect transcription regulation However, these bind to regulatory proteins and not to DNA directly In some cases, the presence of a small effector molecule may increase transcription These molecules are termed inducers They function in two ways Bind activators and cause them to bind to DNA Bind repressors and prevent them from binding to DNA Genes that are regulated in this manner are termed inducible In other cases, the presence of a small effector molecule may inhibit transcription Corepressors bind to repressors and cause them to bind to DNA Inhibitors bind to activators and prevent them from binding to DNA Genes that are regulated in this manner are termed repressible 14-6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

14-7 Regulatory proteins have two binding sites Figure 14.2 One for a small effector molecule The other for DNA Figure 14.2 14-7

Figure 14.2 14-8

The Phenomenon of Enzyme Adaptation At the turn of the 20th century, scientists made the following observation A particular enzyme appears in the cell only after the cell has been exposed to the enzyme’s substrate This observation became known as enzyme adaptation François Jacob and Jacques Monod at the Pasteur Institute in Paris were interested in this phenomenon They focused their attention on lactose metabolism in E. coli to investigate this problem 14-9 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The lac Operon An operon is a regulatory unit consisting of a few structural genes under the control of one promoter It encodes polycistronic mRNA that contains the coding sequence for two or more structural genes This allows a bacterium to coordinately regulate a group of genes that encode proteins with a common function An operon contains several different regions Promoter; terminator; structural genes; operator 14-10 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

There are two distinct transcriptional units 1. The actual lac operon Figure 14.3a shows the organization and transcriptional regulation of the lac operon genes There are two distinct transcriptional units 1. The actual lac operon a. DNA elements Promoter  Binds RNA polymerase Operator  Binds the lac repressor protein CAP site  Binds the Catabolite Activator Protein (CAP) b. Structural genes lacZ  Encodes b-galactosidase Enzymatically cleaves lactose and lactose analogues Also converts lactose into allolactose (an isomer) lacY  Encodes lactose permease Membrane protein required for transport of lactose and analogues lacA  Encodes transacetylase Covalently modifies lactose and analogues Its functional necessity remains unclear 14-11 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

There are two distinct transcriptional units Figure 14.3a shows the organization and transcriptional regulation of the lac operon genes There are two distinct transcriptional units 2. The lacI gene Not considered part of the lac operon Has its own promoter, the i promoter Constitutively expressed at fairly low levels Encodes the lac repressor The lac repressor protein functions as a tetramer Only a small amount of protein is needed to repress the lac operon There is usually ten tetramer proteins per cell 14-12 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 14.3 14-13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The lac Operon Is Regulated By a Repressor Protein The lac operon can be transcriptionally regulated 1. By a repressor protein 2. By an activator protein The first method is an inducible, negative control mechanism It involves the lac repressor protein The inducer is allolactose It binds to the lac repressor and inactivates it Refer to Figure 14.4 14-14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

RNA pol cannot access the promoter Constitutive expression The lac operon is now repressed Therefore no allolactose Figure 14.4 14-15 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

14-16 Figure 14.4 Translation The lac operon is now induced The conformation of the repressor is now altered Repressor can no longer bind to operator Some gets converted to allolactose Figure 14.4 14-16 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

14-17 Figure 14.5 The cycle of lac operon induction and repression Repressor does not completely inhibit transcription So very small amounts of the enzymes are made The cycle of lac operon induction and repression Figure 14.5 14-17 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The lac Operon Is Also Regulated By an Activator Protein The lac operon can be transcriptionally regulated in a second way, known as catabolite repression When exposed to both lactose and glucose E. coli uses glucose first, and catabolite repression prevents the use of lactose When glucose is depleted, catabolite repression is alleviated, and the lac operon is expressed The sequential use of two sugars by a bacterium is termed diauxic growth 14-31 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The lac Operon Is Also Regulated By an Activator Protein The small effector molecule in catabolite repression is not glucose This form of genetic regulation involves a small molecule, cyclic AMP (cAMP) It is produced from ATP via the enzyme adenylyl cyclase cAMP binds an activator protein known as the Catabolite Activator Protein (CAP) Also termed the cyclic AMP receptor protein (CRP) 14-32 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The lac Operon Is Also Regulated By an Activator Protein The cAMP-CAP complex is an example of genetic regulation that is inducible and under positive control The cAMP-CAP complex binds to the CAP site near the lac promoter and increases transcription In the presence of glucose, the enzyme adenylyl cyclase is inhibited This decreases the levels of cAMP in the cell Therefore, cAMP is no longer available to bind CAP Transcription rate decreases 14-33 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

14-34 Figure 14.8 (b) Lactose but no cAMP Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 14.8 14-35 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The lac Operon Has Three Operator Sites for the lac Repressor Detailed genetic and crystallographic studies have shown that the binding of the lac repressor is more complex than originally thought In all, three operator sites have been discovered O1  Next to the promoter O2  Downstream in the lacZ coding region O3  Slightly upstream of the CAP site Refer to Figure 14.9 14-36 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

14-37 Figure 14.9 Repression is 1,300 fold Therefore, transcription is 1/1,300 the level when lactose is present No repression ie: Constitutive expression The identification of three lac operator sites Figure 14.9 14-37

The results of Figure 14.9 supported the hypothesis that the lac repressor must bind to two of the three operators to cause repression It can bind to O1 and O2 , or to O1 and O3 But not O2 and O3 If either O2 or O3 is missing maximal repression is not achieved Binding of the lac repressor to two operator sites requires that the DNA form a loop A loop in the DNA brings the operator sites closer together This facilitates the binding of the repressor protein Refer to Figure 4.14 14-38 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Each repressor dimer binds to one operator site Figure 14.10 14-39 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display