Molecular Biology I BIO316 Lecture 8 PDF

Molecular Biology I BIO316 Lecture 8 Regulation of Gene Expression in Prokaryotes Prepared by Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics ...

Molecular Biology I BIO316 Lecture 8 Regulation of Gene Expression in Prokaryotes Prepared by Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  Regulation of gene expression Associate Professor of Biochemistry and Molecular Genetics  Gene expression begins at the promoter, where RNA polymerase binds to initiate transcription.  Gene transcription must be selective.  Two types of regulatory proteins bind to DNA to regulate the gene expression: repressor proteins and activator proteins.  In negative regulation, binding of a repressor protein prevents transcription.  In positive regulation, an activator protein binds DNA to stimulate transcription. (1) Lactose metabolism in E. coli  In the presence of lactose, the concentration of the enzymes responsible for its metabolism increases rapidly.  The enzymes responsible for lactose metabolism are inducible, and lactose serves as the inducer.  Dr. Rami Elshazli (1) Lactose metabolism in E. coli Associate Professor of Biochemistry and Molecular Genetics  An operon is the functioning unit of DNA containing a cluster of genes under the control of a single promoter.  The genes are transcribed together into an mRNA strand and translated together in the cytoplasm.  An operon is made up of several structural genes arranged under a common promoter and regulated by a common operator.  The most famous example of an operon is the lac operon in Escherichia coli (E. coli), which controls the breakdown of lactose.  Operons consist of:  Promoter: A DNA sequence where RNA polymerase binds to initiate transcription.  Regulatory genes: Genes that produce repressor or  Operator: A segment of DNA that acts as a regulatory activator proteins that can bind to the operator. switch, controlled by a repressor protein.  These genes influencing whether the structural genes  Structural genes: The actual genes that code for are expressed or not. proteins.  Dr. Rami Elshazli Structural genes of lac operon Associate Professor of Biochemistry and Molecular Genetics  The lacZ gene encodes β-galactosidase,  The lacY gene specifies the  Genes coding for the primary structure of an enzyme are an enzyme whose primary role is to primary structure of permease, called structural genes. convert the disaccharide lactose to the an enzyme that facilitates the  There are three structural genes in the lac operon. monosaccharide glucose + galactose. entry of lactose into the bacterial cell.  The lacA gene codes for the enzyme transacetylase.  This gene may be involved in the removal of toxic by-products of lactose digestion from the cell.  All three genes are transcribed as a single unit, resulting in a so- called polycistronic mRNA.  Single mRNA is simultaneously translated into all three gene products.  Structural genes of lac operon  The repressor gene of the lac operon is known as lacI.  It plays a crucial role in regulating the expression of the genes within the operon, which are responsible for the metabolism of lactose in E. coli.  The lacI gene is located upstream of the lac operon and encodes the LacI repressor protein.  The repressor protein binds to the operator region, preventing RNA polymerase from transcribing the downstream genes (lacZ, lacY, and lacA), which are responsible for the metabolism of lactose.  When lactose is present, it binds to the repressor protein, causing a conformational change that releases it from the operator, allowing the operon to be transcribed and enabling the metabolism of lactose. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli Structural genes of lac operon Associate Professor of Biochemistry and Molecular Genetics  The lac operon in E. coli enables the bacterium to metabolize lactose when glucose is not available.  Structural Genes:  lacZ: Encodes β-galactosidase, an enzyme that breaks down lactose into glucose and galactose.  lacY: Encodes lactose permease, which transports lactose into the cell.  lacA: Encodes transacetylase, involved in detoxifying certain compounds related to lactose metabolism.  Regulatory Elements:  Promoter: Where RNA polymerase binds to initiate transcription.  Operator: A DNA sequence that acts as the binding site for the repressor protein, controlling whether the operon is active.  Repressor gene (lacI): Located upstream of the operon and transcribed independently, this gene produces the lac repressor protein. The Operon Model: Negative Control  The lac operon consists of the lacZ, lacY, and lacA structural genes, as well as the adjacent sequences of DNA referred to as the operator region.  The lacI gene regulates the transcription of the structural genes by producing a repressor molecule (lacI).  This repressor is allosteric, meaning that the molecule reversibly interacts with another molecule, undergoing both a conformational change in three-dimensional shape and a change in chemical activity.  The repressor normally binds to the DNA sequence of the operator region.  It inhibits the action of RNA polymerase, effectively repressing the transcription of the structural genes. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  The Operon Model: Negative Control  When lactose is present, this sugar binds to the repressor and causes an allosteric (conformational) change.  The change alters the binding site of the repressor, rendering it incapable of interacting with operator DNA.  Because transcription occurs only when the repressor protein fails to bind to the operator region, regulation is said to be under negative control. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics The Operon Model: Negative Control  The lac operon contains a promoter, to which RNA polymerase binds to initiate transcription.  The lac operon contains an operator, to which a repressor protein can bind.  The repressor protein has two binding sites:  One that binds to the operator DNA.  The other that binds to the carbohydrate inducer.  The environmental signal that induces the lac operon is lactose, but the actual inducer is allolactose, a molecule that forms from lactose once it enters the cell.  In the absence of the inducer, the repressor protein fits into the major groove of the operator DNA and binds to a specific nucleotide base sequence.  This prevents the binding of RNA polymerase to the promoter, and the operon is not transcribed. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  The Operon Model: Negative Control  When the inducer is present, it binds to the repressor and changes the shape of the repressor.  This change in conformational structure prevents the repressor from binding to the operator.  As a result, RNA polymerase can bind to the promoter and start transcribing the structural genes of the lac operon. The Catabolite-Activating Protein (CAP)  In the lac operon, the role of β-galactosidase is to cleave lactose into glucose and galactose.  Then, to be used by the cell, the galactose must be converted to glucose.  If the cell found in the environment sufficient amounts of lactose and glucose.  It would not be energetically efficient for a cell to induce transcription of the lac operon. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  The Catabolite-Activating Protein (CAP)  The catabolite-activating protein (CAP), is involved in diminishing the expression of the lac operon when glucose is present.  This inhibition is called catabolite repression.  When the lac repressor is bound to the inducer (lactose), the lac operon is activated, and RNA polymerase transcribes the structural genes.  The transcription is initiated as a result of the binding between RNA polymerase and the promoter region.  In the absence of glucose and under inducible conditions, CAP exerts positive control by binding to the CAP site, facilitating RNA-polymerase binding at the promoter.  For maximal transcription of the structural genes to occur, the repressor must be bound by lactose and CAP must be bound to the CAP-binding site.  Because of its involvement with cAMP, CAP is also called cyclic AMP receptor protein (CRP). Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli The Catabolite-Activating Protein (CAP) Associate Professor of Biochemistry and Molecular Genetics  The presence of glucose could inhibit CAP binding.  Upon binding the cyclic adenosine monophosphate (cAMP) with the CRP, the CAP binding with the promoter is occurred.  The level of cAMP is dependent on an enzyme, adenyl cyclase, which catalyzes the conversion of ATP to cAMP.  Glucose inhibits the activity of adenyl cyclase, causing a decline in the level of cAMP in the cell.  Under this condition, CAP cannot form the cAMP–CAP complex essential to the positive control of transcription of the lac operon. The Catabolite-Activating Protein (CAP)  The lac operon is positively regulated by an activator called the catabolite activator protein (CAP).  CAP is controlled by a small effector molecule, cyclic AMP (cAMP), which is produced from ATP via an enzyme known as adenylyl cyclase.  When cAMP binds to CAP, the cAMP-CAP complex binds to the CAP site near the lac promoter.  This causes a bend in the DNA that enhances the ability of RNA polymerase to bind to the promoter.  The presence of glucose in the environment inhibits the production of cAMP, thereby preventing the binding of CAP to the DNA.  In this way, glucose blocks the activation of the lac operon, thereby inhibiting transcription. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli The Catabolite-Activating Protein (CAP) Associate Professor of Biochemistry and Molecular Genetics  The four possible environmental conditions that an E. coli bacterium might experience with the presence of the two sugars lactose and glucose.  High lactose and high glucose: The rate of transcription of the lac operon is low because CAP does not activate transcription.  Under these conditions, the bacterium primarily uses glucose rather than lactose.  The bacterium conserves energy by using one type of sugar at a time.  High lactose and low glucose: The transcription rate of the lac operon is very high because CAP is bound to the CAP site and lac repressor is not bound to the operator.  Low lactose and high or low glucose: When lactose levels are low, lac  Under these conditions, the bacterium readily repressor prevents transcription of the lac operon, whether glucose metabolizes lactose. levels are high or low. (2) Tryptophan metabolism in E. coli  This model is an example of the repressible operon.  If tryptophan is present in sufficient quantity in the growth medium of E. coli, the tryptophan synthetase enzyme is not produced.  It is energetically advantageous for bacteria to repress expression of genes involved in tryptophan synthesis when tryptophan is present in the growth medium.  Five contiguous genes encoded a series of enzymes on the E. coli chromosome are involved in tryptophan synthesis.  These genes are part of the tryptophan operon.  In the presence of tryptophan, they are coordinately repressed and none of the enzymes are produced. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics (2) Tryptophan metabolism in E. coli Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  The presence of an inactive repressor alone cannot interact with the operator region of the operon.  The repressor is an allosteric molecule that can bind to tryptophan.  When tryptophan is present, the resultant complex of repressor and tryptophan attains a conformational change that binds to the operator, repressing transcription.  The trp operon is a set of genes that when transcribed together encode the enzymes that cause bacteria to generate the amino acid tryptophan.  The trp operon was initially defined in Escherichia coli.  When tryptophan is available in the environment, the genes for tryptophan synthesis are inhibited via regulation of the operon.  Tryptophan (Trp) Operon  The E. coli trp operon is a cluster of genes that code for biosynthetic enzymes for the amino acid tryptophan.  When tryptophan levels are low, the trp operon is expressed (turned “on”).  When tryptophan levels are high, the trp operon is repressed (turned “off”).  The trp repressor controls the trp operon.  When coupled to tryptophan, the trp repressor inhibits operon expression.  Bacteria such as Escherichia coli require amino acids to exist because they must construct proteins.  Tryptophan is one of the amino acids they require. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Tryptophan (Trp) Operon  If tryptophan is present in the environment, E. coli will utilize it to produce proteins.  E. coli can also produce tryptophan using enzymes expressed by five genes.  If tryptophan is present in the environment, E. coli bacteria do not need to generate it and transcription of the trp operon genes is “turned off.”  If tryptophan is scarce, the operon is activated and the genes are transcribed, and more tryptophan is created.  This operon exemplifies repressible negative gene expression regulation.  The trp operon contains five structural genes that encode the enzymes required to produce tryptophan: trpE, trpD, trpC, trpB, and trpA. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli Structure of the trp operon Associate Professor of Biochemistry and Molecular Genetics  The trp operon consists of five genes encoding enzymes required for tryptophan production,  Additionally, it possesses a promoter (RNA polymerase binding site) and an operator (binding site for a repressor protein).  The trp operon’s genes are transcribed as a single mRNA.  Dr. Rami Elshazli Structure of the trp operon Associate Professor of Biochemistry and Molecular Genetics  Five structural genes comprise the Trp operon.  TrpE: Encodes the Anthranilate synthase enzyme I.  TrpD: Encodes the Anthranilate synthase enzyme II.  TrpC: Encodes the enzyme N-5’-Phosphoribosyl anthranilate isomerase and Indole-3- glycerolphosphate synthase.  TrpB: Encodes the enzyme tryptophan synthase-B subunit.  TrpA: Encodes the enzyme tryptophan synthase-A subunit.  (2) Tryptophan (Trp) Operon  The trp operon contains the repressive regulator gene trpR.  When tryptophan is present, the trpR protein binds to the operator, preventing RNA polymerase from transcribing the trp operon.  This operon possesses two levels of regulation:  Repression: works at the transcription initiation level.  Attenuation: works at the transcription termination level. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics