Activation & Repression Jacob and Monod in 1961 described their operon model in a classic paper. Their hypothesis was to a large extent based on observations on the regulation of lactose metabolism by the intestinal bacterium E. coli. The molecular mechanisms responsible for the regulation of the genes involved in the metabolism of lactose are now among the best-understood in any organism. β-Galactosidase hydrolyzes the β-galactoside lactose to galactose and glucose. The gene encoding β-galactosidase (lacZ) is clustered with the genes encoding lactose permease (lacY) and thiogalactoside transacetylase (lacA). The genes encoding these three enzymes, along with the lac promoter and lac operator (a regulatory region), and the lacI gene encoding the LacI repressor are physically linked and constitute the lac operon as depicted in Figure 1.

Fig1. The positional relationships of the protein coding and regulatory elements of the ~6kbp lac operon. lacZ encodes β-galactosidase, lacY encodes a permease, and lacA encodes a transacetylase. lacI encodes the lac operon repressor protein. Also shown is the transcription start site for lac operon transcription (TSS). Note that the binding site for the LacI protein (ie, lac repressor)—the lac operator (Operator)—overlaps the lac promoter. Immediately upstream of the lac operon promoter is the binding site (CRE) for the cAMP-binding protein, CAP, the positive regulator of lac operon transcription. See Figure 38–3 for more detail.

This genetic arrangement of the lac operon allows for coordinate expression of the three enzymes concerned with lactose metabolism. Each of the linked operon genes is transcribed into one large polycistronic mRNA molecule that contains multiple independent translation start (AUG) and stop (UAA) codons for each of the three cistrons. Thus, each protein is translated separately, and they are not processed from a single large precursor protein.

It is now conventional to consider that a gene includes regulatory sequences as well as the region that encodes the primary transcript. Although there are many historical exceptions, a gene is generally italicized in lower case and the encoded protein, when abbreviated, is expressed in roman type with the first letter capitalized. For example, the gene lacI encodes the repressor protein LacI. When E. coli is presented with lactose or some specific lactose analogs under appropriate nonrepressing conditions (eg, high concentrations of lactose, no or very low glucose in media; see following discussion), the expression of the activities of β-galactosidase, galactoside permease, and thiogalactoside transacetylase is increased 100-fold to 1000-fold. This is a type A response. The kinetics of induction can be quite rapid ;lac-specific mRNAs are fully induced within ~5 minutes after addition of lactose to a culture; β-galactosidase protein is maximal within 10 minutes. Under fully induced conditions, there can be up to 5000 β-galactosidase molecules per cell, an amount about 1000 times greater than the basal, uninduced level. Upon removal of the signal, that is, the inducer, the syn thesis of these three enzymes declines.

When E. coli is exposed to both lactose and glucose as sources of carbon, the cells first metabolize the glucose and then temporarily stop growing until the genes of the lac operon become induced to provide the ability to metabolize lactose as a usable energy source. Although lactose is present from the beginning of the bacterial growth phase, the cell does not induce those enzymes necessary for catabolism of lactose until glucose has been exhausted. This phenomenon was first thought to be attributable to repression of the lac operon by some catabolite of glucose; hence, it was termed catabolite repression. It is now known that catabolite repression is in fact mediated by a catabolite activator protein (CAP) in conjunction 3′,5′ cyclic Adenosine monophosphate (cAMP). This protein is also referred to as the cAMP regulatory protein (CRP). The expression of many inducible enzyme systems or operons in E. coli and other prokaryotes is sensitive to catabolite repression, as discussed in following discussion.

The physiology of induction of the lac operon is well understood at the molecular level (Figure 2). Expression of the normal lacI gene of the lac operon is constitutive; it is expressed at a constant rate, resulting in formation of the sub units of the lac repressor. Four identical subunits with molecular weights of 38,000 assemble into a tetrameric Lac repressor molecule. The LacI repressor protein molecule, the product of lacI, has a very high affinity (dissociation constant, Kd about 10⁻¹³ mol/L) for the operator locus. The operator locus is a region of double-stranded DNA that exhibits a twofold rotational symmetry and an inverted palindrome (indicated by arrows about the dotted axis) in a region that is 21-bp long, shown as follows:

Fig2. The mechanism of repression, derepression, and activation of thelac operon. When no inducer is present (A), the constitutively synthesized lacI gene products form a repressor tetramer that binds to the operator. Repressor-operator binding prevents the binding of RNA polymerase and consequently prevents transcription of the lacZ, lacY, and lacA genes into a polycistronic mRNA. When inducer is present, but glucose is also present in the culture medium (B), the tetrameric repressor molecules are conformationally altered by inducer, and cannot efficiently bind to the operator locus (affinity of binding reduced >1000-fold). However, RNA polymerase will not efficiently bind the promoter and initiate transcription because positive protein–protein interactions between CRE-bound CAP protein and RNA polymerase fail to occur; thus, the lac operon is not efficiently transcribed. However, when inducer is present, and glucose is depleted from the medium (C), adenylyl cyclase is activated and cAMP is produced. This cAMP binds with high affinity to its binding protein the cyclic AMP activator protein, or CAP. The CAP-cAMP complex binds to its recognition sequence (CRE, the cAMP response element) at lac operon nucleotide coordinate −50. Direct protein–protein contacts between the CRE-bound CAP and the RNA polymerase increases promoter binding more than 20-fold; hence RNAP will efficiently transcribe the lac operon and the polycistronic lacZ-lacY-lacA mRNA molecule formed can be translated into the corresponding protein molecules β-galactosidase, permease, and transacetylase as shown. This protein production enables cellular catabolism of lactose as the sole carbon source for growth.

At any one time, only two of the four subunits of the repressor appear to bind to the operator; within the 21-base-pair operator region nearly every base of each base pair is involved in LacI recognition and binding. Binding occurs mostly in the major groove without interrupting the base-paired, double helical nature of the operator DNA. The operator locus (ie, LacI binding site) is between the promoter, the site where the DNA-dependent RNA polymerase attaches to commence transcription, and the transcription initiation site of the lacZ gene, the structural gene for β-galactosidase (see Figures 1 ). When bound to the operator locus, the LacI repressor molecule prevents transcription of the distal structural genes, lacZ, lacY, and lacA by interfering with the binding of RNA polymerase to the promoter; RNA polymerase and LacI repressor cannot be effectively bound to the lac operon at the same time. Thus, the LacI repressor molecule is a negative regulator, and in its presence (and in the absence of inducer; see following discussion), expression from the lacZ, lacY, and lacA genes is very, very low. There are normally about 30 repressor tetramer molecules in the cell, a concentration (3 × 10⁻⁸ mol/L) of tetramer sufficient to effect, at any given time, more than 95% occupancy of the one lac operator element in a bacterium, thus ensuring low (but not zero) basal lac operon gene transcription in the absence of inducing signals.

A lactose analog that is capable of inducing the lac operon while not itself serving as a substrate for β-galactosidase is an example of a gratuitous inducer. An example is isopropyl thiogalactoside (IPTG). The addition of lactose or of a gratuitous inducer such as IPTG to bacteria growing on a poorly utilized carbon source (such as succinate) results in prompt induction of the lac operon enzymes. Small amounts of the gratuitous inducer or of lactose are able to enter the cell even in the absence of permease. The LacI repressor molecules— both those attached to the operator loci and those free in the cytosol—have a high affinity for the inducer. Binding of the inducer to repressor molecule induces a conformational change in the structure of the repressor that causes a decrease in operator DNA occupancy because its affinity for the operator is now 104 times lower (Kd about 10⁻⁹ mol/L) than that of LacI in the absence of IPTG. DNA-dependent RNA polymerase can now more efficiently compete with LacI and bind to the promoter, and transcription will begin, although this process is relatively inefficient (see following discussion). In such a manner, an inducer derepresses the lac operon and allows transcription of the genes encoding β-galactosidase, galactoside permease, and thiogalactoside transacetylase. Translation of the polycistronic mRNA can occur even before transcription is completed. Derepression of the lac operon allows the cell to synthesize the enzymes necessary to catabolize lactose as an energy source. Based on the physiology just described, IPTG induced expression of transfected plasmids bearing the lac operator–promoter ligated to appropriate bioengineered con structs is commonly used to express mammalian recombinant proteins in E. coli.

In order for the RNA polymerase to form a PIC at the promoter site most efficiently, the cAMP-CAP complex must also be present in the cell. By an independent mechanism, the bacterium accumulates cAMP only when it is starved for a source of carbon. In the presence of glucose—or of glycerol in concentrations sufficient for growth—the bacteria will lack sufficient cAMP to bind to CAP because glucose inhibits adenylyl cyclase, the enzyme that converts ATP to cAMP. Thus, in the presence of glucose or glycerol, cAMP saturated CAP is lacking, so that the DNA-dependent RNA polymerase cannot initiate transcription of the lac operon at the maximal rate. However, in the presence of the CAP-cAMP complex, which binds to CAP Response Element (CRE) DNA just upstream of the promoter site, transcription occurs at maximal levels (see Figure 2). Studies indicate that a region of CAP directly contacts the RNA polymerase (RNAP) α subunit, and these protein–protein interactions facilitate the binding of RNAP to the promoter. Thus, the CAP-cAMP regulator is acting as a positive regulator because its presence is required for optimal gene expression. The lac operon is therefore controlled by two distinct, ligand-modulated DNA binding trans-factors; one that acts positively (cAMP-CRP complex) to facilitate productive binding of RNA polymerase to the promoter and one that acts negatively (LacI repressor) that antagonizes RNA polymerase promoter binding. Maximal activity of the lac operon occurs when glucose levels are low (high cAMP with CAP activation) and lactose is present (LacI is prevented from binding to the operator) as shown in Figure 2, panel C.

With the above information in hand, it becomes relatively to predict the effects of mutations in various components of the lac-system upon lac operon expression. When the lacI gene has been mutated so that its product, LacI, is not capable of binding to operator DNA, the organism will exhibit constitutive expression of the lac operon. In a contrary manner, an organism with a lacI gene mutation that produces a LacI protein which prevents the binding of lactose or other small molecule inducer to the repressor will remain repressed even in the presence of the inducer molecule, because such ligands cannot bind to the repressor on the operator locus in order to derepress the operon. Similarly, bacteria harboring mutations in their lac operator locus such that the operator sequence will not bind a normal repressor molecule will constitutively express the lac operon genes. Mechanisms of positive and negative regulation comparable to those described here for the lac system have been observed in eukaryotic cells.

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Biologic Systems Exhibit Three Types of Temporal Responses to a Regulatory Signal

Regulated Expression of Genes is Required for Development, Differentiation, & Adaptation

Posttranslational Processing Affects the Activity of many Proteins

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