The operon concept describes how transcription is controlled in bacteria through interactions between trans-acting regulatory proteins and cis-acting DNA sequences. The lac operon consists of three structural genes (lacZ, lacY, lacA) that are transcribed together and encode enzymes for lactose metabolism. Expression of these genes is negatively regulated by the LacI repressor protein, which binds to the lac operon's operator region and prevents transcription unless lactose or allolactose are present to induce expression.
The operon concept describes how transcription is controlled in bacteria through interactions between trans-acting regulatory proteins and cis-acting DNA sequences. The lac operon consists of three structural genes (lacZ, lacY, lacA) that are transcribed together and encode enzymes for lactose metabolism. Expression of these genes is negatively regulated by the LacI repressor protein, which binds to the lac operon's operator region and prevents transcription unless lactose or allolactose are present to induce expression.
The operon concept describes how transcription is controlled in bacteria through interactions between trans-acting regulatory proteins and cis-acting DNA sequences. The lac operon consists of three structural genes (lacZ, lacY, lacA) that are transcribed together and encode enzymes for lactose metabolism. Expression of these genes is negatively regulated by the LacI repressor protein, which binds to the lac operon's operator region and prevents transcription unless lactose or allolactose are present to induce expression.
• The basic concept for the way transcription is controlled in bacteria • Proposed by Francois Jacob and Jacques Monod in 1961 • Distinguished between two types of sequences in DNA • Sequences that code for trans- acting products (usually proteins) • Cis-acting DNA sequences • Gene activity is regulated by the specific interactions of the trans-acting products with the cis-acting sequences Gene • A sequence of DNA that codes for a diffusible product • Either RNA or protein • The product diffuses away from its site of synthesis to act elsewhere • Any gene product that is free to diffuse to find its target is described as trans-acting • Cis-acting applies to any sequence of DNA that functions exclusively as a DNA sequence • Affecting only the DNA to which it is physically linked Components of genes and regulatory circuits • Structural gene and regulator gene • A structural gene is simply any gene that codes for a protein (or RNA) product • Protein structural genes represent an enormous variety of structures and functions, including structural proteins, enzymes with catalytic activities and regulatory proteins • A type of structural gene is a regulator gene • Which simply describes a gene that codes for a protein or an RNA involved in regulating the expression of other genes Components of genes and regulatory circuits • A regulator gene codes for a protein that controls transcription by binding to particular site(s) on DNA • This interaction can regulate a target gene in: • Positive manner (the interaction turns the gene on) • Negative manner (the interaction turns the gene off) • The sites on DNA are usually located just upstream (but not exclusively) of the target gene Transcription Unit • The sequences that mark the beginning and end of the transcription unit- promoter and terminator (cis-acting sites) • A promoter serves to initiate transcription only of the gene(s) physically collected to it on the same stretch of DNA • In the same way, a terminator can terminate transcription only by an RNA polymerase that has traversed the preceding gene(s) • In their simplest forms, promoters and terminators are cis- acting elements that are recognized by the same trans- acting species (by RNAP) Negative control • Transcription control in bacteria • A repressor protein prevents a gene from being expressed • Absence of the negative regulator, the gene is expressed • Operator (cis-acting site) close to the promoter, binding site for the repressor protein • When the repressor binds to the operator • RNA polymerase is prevented from initiating transcription • Gene expression is turned off Negative control
• Trans-acting repressor binds to the
cis-acting operator • Turn off transcription Positive control • An alternative mode of control is positive control • In bacteria (probably) equal frequency to negative control • Most common mode of control in eukaryotes • A transcription factor is required to assist RNAP in initiation at the promoter • Absence of the positive regulator, the gene is inactive • RNAP cannot by itself initiate transcription at the promoter Positive control
• Trans-acting factor must
bind to the cis-acting site • In order for RNAP to initiate transcription at the promoter Inducible gene • A gene encodes an enzyme may be regulated by the conc. of its substrate or product (or a chemical derivative of either) • Bacteria need to respond swiftly to changes in their environment • Fluctuations in the supply of nutrients (sugars glucose or lactose) can occur at any time • Survival depends on the ability to switch from metabolizing one substrate to another • Economies are vital • A bacterium indulges in energetically expensive ways to meet the demands of the environment is at disadvantage Inducible gene • Bacterium avoids synthesizing the enzymes of a pathway in the absence of the substrate • But, ready to produce the enzymes, if the substrate appear • The synthesis of enzymes in response to the appearance of a specific substrate • Induction • The gene is an inducible gene Repression • The opposite of induction is repression • Where the repressible gene is controlled by the amount of the product made by the enzyme • In E. coli, synthesizes the AA tryptophan by an enzyme complex containing tryptophan synthetase and four enzymes • If, however, tryptophan is provided in the medium on which the bacteria are growing • the production of the enzyme is immediately halted • This allows the bacterium to avoid devoting its resources to unnecessary synthetic activities Induction and repression • Represent similar phenomena • In one case the bacterium adjusts its ability to use a given substrate (such as lactose) for growth • In the other it adjusts its ability to synthesize a particular metabolic intermediate (such as an essential amino acid) • The trigger for either type of adjustment is a small molecule that is: • the substrate (or related to the substrate) for the enzyme • or the product of the enzyme activity Inducer and corepressors • Small molecules that cause the production of enzymes • That are able to metabolize them (or their analogues) • Inducers • Those that prevent the production of enzymes • That are able to synthesize them • Corepressors Two ways of looking at regulation • Negative versus positive control • inducible versus repressible control • Are combined to give four different patterns of gene regulation • Negative inducible • Negative repressible • Positive inducible • Positive repressible Structural Gene Clusters are Coordinately Controlled • Bacterial genes are organized into operons • Include genes that code for proteins whose functions are related • Genes coding for the enzymes of a metabolic pathway are commonly organized into a cluster lac operon • Cluster of the lac operon contains 3 lac structural genes • lacZ, lacY and lacA • Structural gene cluster is transcribed into a single polycistronic mRNA from a promoter • Where initiation of transcription is regulated • The lac operon occupies -6000 bp of DNA Long lacZ gene starts at base 39 At the left the lac I gene has: lacY Promoter lacA Terminator terminator
End of the lacI region is Operator, O, occupies first 26 bp of the
adjacent to lacZYA promoter, P transcription unit The protein products enable cells to take up and metabolize β -galactoside sugars, such as lactose The roles of the three structural genes • lacZ codes for the enzyme β -galactosidase • Active form is a tetramer of -500 kD • The enzyme breaks the complex β -galactoside into its component sugars: Lactose • Into glucose and galactose • This enzyme also produces an important by product, β -1,6- allolactase The roles of the three structural genes • lacY codes for the β -galactoside permease • 30-kD membrane-bound protein constituent of the transport system • Transports β -galactosides into the cell • lacA codes for β -galactoside transacetylase • Enzyme that transfers an acetyl group • From acetyl-CoA to β-galactosides The roles of the three structural genes • Mutations in either lacZ or lacY can create the lac genotype, in which cells cannot utilize lactose • The lacZ mutations abolish enzyme activity • Directly preventing metabolism of lactose • The lacY mutants cannot take up lactose efficiently from the medium The roles of the three structural genes • The entire system • including structural genes • the elements that control their expression • forms a common unit of regulation • an operon • The activity of the operon is controlled by regulator gene(s) whose protein products interact with the cis-acting control elements The lac Operon: Negative Inducible • Transcription of the lacZYA genes is controlled by a regulator protein encoded by the LacI gene • lacI comprises an independent transcription unit with its own promoter and terminator • lacI need not be located near the structural genes because it specifies a diffusible product • The lacI gene can function equally well if moved elsewhere, or can be carried on a separate DNA molecule • A trans-acting regulator The lac Operon: Negative Inducible • The lacZYA genes are negatively regulated • Transcribed, unless turned off by the regulator protein • Repression is not an absolute phenomenon • Turning off a gene is not like turning off a lightbulb • Repression can be reduction in transcription by 5 or 100-fold • A mutation that inactivates the regulator causes the structural genes to be continually expressed, constitutive expression • Product of lacI- lac repressor • Its function is to prevent the expression of the lacZYA structural genes The lac Operon: Negative Inducible • Repressor is a tetramer of identical SUs of 38 kD each • A wild-type cell contains ~ 10 tetramers • Repressor gene is not controlled; it is an unregulated gene • Transcribed into a monocistronic mRNA • At a rate that appears to be governed simply by the affinity of its (poor) promoter for RNA polymerase The lac Operon: Negative Inducible • lacI is transcribed into a poor mRNA • This is a common way to restrict the amount of protein made • In this case, the mRNA has no 5 ' UTR • Which restricts the ability of a ribosome to start translation • These two features account for the low abundance of lac repressor protein in the cell The lac Operon: Negative Inducible • Repressor functions by binding to an operator (Olac) at the start of the lacZYA cluster • Sequence of the operator includes an inverted repeat • The operator lies b/w the promoter (Plac) and the structural genes (lacZYA) • When the repressor binds at the operator • Prevents RNAP polymerase from initiating transcription at the promoter The lac Operon: Negative Inducible • Operator extends from position -5 to +21 upstream of the mRNA start point within the transcription unit • Overlaps the 3', right end of the promoter •A mutation that inactivates the operator • Causes constitutive expression The lac Operon: Negative Inducible • E. coli are grown in the absence of β-galactoside- no need for β -galactosidase • Contain very few molecules of the enzyme, about 5/cell • Suitable substrate added, the enzyme activity appears very rapidly in the bacteria • Within 2-3 minutes, bacterium accumulates ~5000 molecules of enzyme • Under suitable conditions, β -galactosidase can account for 5-10% of the total soluble protein of the bacterium • Substrate removed from medium, the synthesis of the enzyme stops as rapidly as it started Essential features of this induction • Control of transcription of the lac operon responds very rapidly to the inducer • In the absence of inducer, the operon is transcribed at a very low basal level • Transcription is stimulated as soon as inducer is added • The amount of lac mRNA increases rapidly to an induced level • Reflects a balance between synthesis and degradation of the mRNA Addition of inducer results in rapid induction of lac mRNA Removal of inducer, rapid cessation of A short lag by synthesis synthesis of the enzymes Lac Repressor Controlled by a Small-Molecule Inducer • The ability to act as inducer or corepressor is highly specific • Only the substrate/product of the regulated enzymes or • a closely related molecule can serve this function • For lac system the natural inducer is not lactose • A byproduct of the LacZ enzyme, allolactose • Also a substrate of LacZ enzyme, does not persist in the cell • Inducers resemble the natural inducers of the lac operon but cannot be metabolized by the enzyme • Isopropylthiogalactoside (IPTG), an inducer • not metabolized by β-galactosidase Lac Repressor Controlled by a Small- Molecule Inducer • Gratuitous inducers: Molecules that induce enzyme synthesis but are not metabolized • Component that represses the lac operon is the lac repressor protein, encoded by lacI gene • The lac repressor protein is induced by allolactose and IPTG to allow expression of lacZYA • The LacZ enzyme (β -galactosidase) utilizes allolactose and lactose as substrates • lacI is not induced by lactose • LacZ enzyme does not metabolize IPTG Lac Repressor Controlled by a Small-Molecule Inducer • The component that responds to the inducer is the repressor protein encoded by lacI • Its target, the lacZYA structural genes, • Transcribed into a single mRNA from the promoter just upstream of lacZ. • State of the repressor determines whether this promoter is turned off or on Lac Repressor Controlled by a Small-Molecule Inducer • In the absence of an inducer, the genes are not transcribed • In an active form, repressor protein is bound to the operator • lac repressor maintains the lac operon in the inactive condition by binding to the operator Lac Repressor Controlled by a Small-Molecule Inducer • Inducer added, repressor is converted into a form that leaves the operator • Addition of inducer converts repressor to a form with low affinity for the operator • Allows RNAP to initiate transcription • Transcription starts at the promoter and proceeds through the genes to a terminator located beyond the 3' end of lacA Lac Repressor Controlled by a Small-Molecule Inducer • Crucial features reside in the dual properties of the repressor: • Can prevent transcription • Can recognize the small-molecule inducer • The repressor has two types of binding site: • One type for the operator DNA • One type for the inducer Lac Repressor Controlled by a Small-Molecule Inducer • When the inducer binds at its site • It changes the structure of the protein to influence the activity of the operator-binding site • The ability of one site in the protein to control the activity of another • Allosteric control