Gene Regulation in Bacteria

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 continuously necessary for the survival of the organism The benefit of regulating genes is that encoded proteins will be produced only when required Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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

TRANSCRIPTIONAL REGULATION The most common way to regulate gene expression in bacteria is at the transcription initiation 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Regulatory proteins have two binding sites One for a small effector molecule The other for DNA

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Activator/Repressor and the effector molecules

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

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

Organization and transcriptional regulation of the lac operon genes There are two distinct transcriptional units 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 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 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The lac operon is now induced Some gets converted to allolactose Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 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 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) 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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The trp Operon The trp operon (pronounced “trip”) is involved in the biosynthesis of the amino acid tryptophan The genes trpE, trpD, trpC, trpB and trpA encode enzymes involved in tryptophan biosynthesis The genes trpR and trpL are involved in regulation trpR  Encodes the trp repressor protein Functions in repression trpL  Encodes a short peptide called the Leader peptide Functions in attenuation Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

RNA pol can bind to the promoter Cannot bind to the operator site Organization of the trp operon and regulation via the trp repressor protein

Organization of the trp operon and regulation via the trp repressor protein

Organization of the trp operon and regulation via the trp repressor protein

The first gene in the trp operon is trpL Attenuation occurs in bacteria because of the coupling of transcription and translation During attenuation, transcription actually begins but it is terminated before the entire mRNA is made A segment of DNA, termed the attenuator, is important in facilitating this termination In the case of the trp operon, transcription terminates shortly past the trpL region Thus attenuation inhibits the further production of tryptophan The segment of trp operon immediately downstream from the operator site plays a critical role in attenuation The first gene in the trp operon is trpL It encodes a short peptide termed the Leader peptide Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Region 2 is complementary to regions 1 and 3 Therefore several stem-loops structures are possible The 3-4 stem loop is followed by a sequence of Uracils It acts as an intrinsic (r-independent) terminator These two codons provide a way to sense if there is sufficient tryptophan for translation Sequence of the trpL mRNA produced during attenuation

There are three possible scenarios Therefore, the formation of the 3-4 stem-loop causes RNA pol to terminate transcription at the end of the trpL gene Conditions that favor the formation of the 3-4 stem-loop rely on the translation of the trpL mRNA There are three possible scenarios 1. No translation 2. Low levels of tryptophan 3. High levels of tryptophan Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Transcription terminates just past the trpL gene Most stable form of mRNA occurs Therefore no ribosome associated with the RNA Possible stem-loop structures formed from trpL mRNA under different conditions of translation

Region 1 is blocked 3-4 stem-loop does not form RNA pol transcribes rest of operon Insufficient amounts of tRNAtrp Possible stem-loop structures formed from trpL mRNA under different conditions of translation

Sufficient amounts of tRNAtrp 3-4 stem-loop forms Translation of the trpL mRNA progresses until stop codon Transcription terminates Ribosome pauses on 2 Region 2 cannot base pair with any other region Possible stem-loop structures formed from trpL mRNA under different conditions of translation

Inducible vs Repressible Regulation The study of many operons revealed a general trend concerning inducible versus repressible regulation Operons involved in catabolism (i.e. breakdown of a substance) are typically inducible The substance to be broken down (or a related compound) acts as the inducer Operons involved in anabolism (ie. biosynthesis of a substance) are typically repressible The inhibitor or corepressor is the small molecule that is the product of the operon Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

TRANSLATIONAL AND POSTTRANSLATIONAL REGULATION Genetic regulation in bacteria is exercised predominantly at the level of transcription However, there are many examples of regulation that occur at a later stage in gene expression For example, regulation of gene expression can be Translational Posttranslational Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Translational Regulation For some bacterial genes, the translation of mRNA is regulated by the binding of proteins A translational regulatory protein recognizes sequences within the mRNA In most cases, these proteins act to inhibit translation These are known as translational repressors Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Translational Regulation Translational repressors inhibit translation in one of two ways 1. Binding next to the Shine-Dalgarno sequence and/or the start codon This will sterically hinder the ribosome from initiating translation 2. Binding outside the Shine-Dalgarno/start codon region They stabilize an mRNA secondary structure that prevents initiation Translational repression is also found in eukaryotes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Translational Regulation A second way to regulate translation is via the synthesis of antisense RNA An RNA strand that is complementary to mRNA Consider, for example, the trait of osmoregulation The ability to control the amount of water inside the cell Must be regulated to prevent shrinkage or lysis The protein ompF in E. coli is important in osmoregulation This outer membrane protein is encoded by the ompF gene OmpF protein is preferentially produced at low osmolarity At high osmolarity its synthesis is decreased Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Its translation is now blocked The expression of another gene, termed micF, is responsible for inhibiting the ompF gene at high osmolarity micF RNA does not code for a protein It is, however, complementary to ompF mRNA It is thus termed antisense RNA Its translation is now blocked Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Posttranslational Regulation A common mechanism to regulate the activity of metabolic enzymes is feedback inhibition The final product in a pathway often can inhibit the an enzyme that acts early in the pathway Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Catalytic site  binds substrate Enzyme 1 is an allosteric enzyme, which means it contains two different binding sites Catalytic site  binds substrate Regulatory site  binds final product of the pathway If the concentration of product 3 becomes high It will bind to enzyme 1 Thereby inhibiting its ability to convert substrate 1 into product 1 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Posttranslational Regulation A second strategy to control the function of proteins is by the covalent modification of their structure Some modifications are irreversible Proteolytic processing Attachment of prosthetic groups, sugars, or lipids Other modifications are reversible and transiently affect protein function Phosphorylation (–PO4) Acetylation (–COCH3) Methylation (–CH3) Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Allosteric protein