Analysis of Lactose Metabolism in E coli Led to the Operon Hypothesis

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. P-Galactosidase hydrolyzes the P-galactoside lactose to galactose and glucose. The structural gene for P-galac-tosidase (lacZ) is clustered with the genes responsible for the permeation of galactose into the cell (lacY) and for thiogalactoside transacetylase (lacA). The structural genes for these three enzymes, along with the lac promoter and lac operator (a regulatory region), are physically associated to constitute the lac operon as depicted in Figure 39-2. This genetic arrangement of the structural genes and their regulatory genes allows for coordinate expression of the three enzymes concerned with lactose metabolism. Each of these linked genes is transcribed into one large mRNA molecule that contains multiple independent translation start (AUG) and stop (UAA) codons for each cistron. Thus, each protein is translated separately, and they are not processed from a single large precursor protein. This type of mRNA molecule is called a polycistronic mRNA. Polycistronic mRNAs are predominantly found in prokaryotic organisms.

It is now conventional to consider that a gene includes regulatory sequences as well as the region that

Promoter Operator siteN

lacI

lacZ

lacY lacA

lac operon

Figure 39-2. The positional relationships of the structural and regulatory genes of the lac operon. lacZ encodes p-galactosidase, lacY encodes a permease, and lacA encodes a thiogalactoside transacetylase. lacI encodes the lac operon repressor protein.

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 non-repressing conditions (eg, high concentrations of lactose, no or very low glucose in media; see below), the expression of the activities of P-galactosidase, galactoside permease, and thiogalactoside transacetylase is increased 100-fold to 1000-fold. This is a type A response, as depicted in Figure 39-1. The kinetics of induction can be quite rapid; lac-specific mRNAs are fully induced within 5-6 minutes after addition of lactose to a culture; P-galactosidase protein is maximal within 10 minutes. Under fully induced conditions, there can be up to 5000 P-galactosidase molecules per cell, an amount about 1000 times greater than the basal, uninduced level. Upon removal of the signal, ie, the inducer, the synthesis of these three enzymes declines.

When E coli is exposed to both lactose and glucose as sources of carbon, the organisms 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 the 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 gene activator protein (CAP) in conjunction with cAMP (Figure 18-5). 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 below.

Operator Promoter I

lac! gene

Operator Promoter I

lac! gene

lacZ gene

RNA polymerase cannot transcribe operator or distal genes (Z,Y, A)

Repressor subunits

Repressor (tetramer)

Repressor subunits

Repressor (tetramer)

lacZ gene

RNA polymerase cannot transcribe operator or distal genes (Z,Y, A)

lacY gene lacA gene

CAP-cAMP

CAP-cAMP

Inactive repressor J^^,

RNA polymerases transcribing genes icmA

icmA

mRNA

Inactive repressor J^^,

RNA polymerases transcribing genes

I Inducers

ß-Galacto- Permease Transacetylase sidase protein protein protein mRNA

ß-Galacto- Permease Transacetylase sidase protein protein protein

Figure 39-3. The mechanism of repression and derepression of the lac operon. When either no inducer is present or inducer is present with glucose (A), the lacI gene products that are synthesized constitutively form a repressor tetramer molecule which binds at the operator locus to prevent the efficient initiation of transcription by RNA polymerase at the promoter locus and thus to prevent the subsequent transcription of the lacZ, lacY, and lacA structural genes. When inducer is present (B), the constitutively expressed lacI gene forms repressor molecules that are conformationally altered by the inducer and cannot efficiently bind to the operator locus (affinity of binding reduced > 1000-fold). In the presence of cAMP and its binding protein (CAP), the RNA polymerase can transcribe the structural genes lacZ, lacY, and lacA, and the polycistronic mRNA molecule formed can be translated into the corresponding protein molecules p-galactosidase, permease, and transacetylase, allowing for the catabolism of lactose.

The physiology of induction of the lac operon is well understood at the molecular level (Figure 39-3). Expression of the normal lacI gene of the lac operon is constitutive; it is expressed at a constant rate, resulting in formation of the subunits of the lac repressor. Four identical subunits with molecular weights of 38,000 assemble into a lac repressor molecule. The LacI repressor protein molecule, the product of lacI, has a high affinity (Kd about 10-13 mol/L) for the operator locus. The operator locus is a region of double-stranded DNA 27 base pairs long with a twofold rotational symmetry and an inverted palindrome (indicated by solid lines about the dotted axis) in a region that is 21 base pairs long, as shown below:

5' - AATTGTGAGCG GATAACAATT 3'- TTA ACACTCG C CTATTGTTAA

The minimum effective size of an operator for LacI repressor binding is 17 base pairs (boldface letters in the above sequence). At any one time, only two subunits of the repressors appear to bind to the operator, and within the 17-base-pair region at least one base of each base pair is involved in LacI recognition and binding. The binding occurs mostly in the major groove without interrupting the base-paired, double-helical nature of the operator DNA. The operator locus is between the promoter site, at which the DNA-dependent RNA polym-erase attaches to commence transcription, and the transcription initiation site of the lacZ gene, the structural gene for P-galactosidase (Figure 39-2). When attached to the operator locus, the LacI repressor molecule prevents transcription of the operator locus as well as of the distal structural genes, lacZ, lacY, and lacA. Thus, the LacI repressor molecule is a negative regulator; in its presence (and in the absence of inducer; see below), expression from the lacZ, lacY, and lacA genes is prevented. There are normally 20-40 repressor tetramer molecules in the cell, a concentration of tetramer sufficient to effect, at any given time, > 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 P-galac-tosidase is an example of a gratuitous inducer. An example is isopropylthiogalactoside (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 in-ducer 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 a repressor molecule attached to the operator locus induces a conformational change in the structure of the repressor and causes it to dissociate from the DNA because its affinity for the operator is now 103 times lower (Kd about 10-9 mol/L) than that of LacI in the absence of IPTG. If DNA-dependent RNA polymerase has already attached to the coding strand at the promoter site, transcription will begin. The polym-erase generates a polycistronic mRNA whose 5' terminal is complementary to the template strand of the operator. In such a manner, an inducer derepresses the lac operon and allows transcription of the structural genes for P-galactosidase, galactoside permease, and thiogalac-toside 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 plas-mids bearing the lac operator-promoter ligated to appro priate bioengineered constructs is commonly used to express mammalian recombinant proteins in E coli.

In order for the RNA polymerase to efficiently form a PIC at the promoter site, there must also be present the catabolite gene activator protein (CAP) to which cAMP is bound. 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 the glucose inhibits adenylyl cyclase, the enzyme that converts ATP to cAMP (see Chapter 42). 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. In the presence of the CAP-cAMP complex, which binds to DNA just upstream of the promoter site, transcription then occurs (Figure 39-3). Studies indicate that a region of CAP contacts the RNA polymerase a subunit and facilitates binding of this enzyme to the promoter. Thus, the CAP-cAMP regulator is acting as a positive regulator because its presence is required for gene expression. The lac operon is therefore controlled by two distinct, ligand-modulated DNA binding trans factors; one that acts positively (cAMP-CRP complex) and one that acts negatively (LacI repressor). 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).

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 an inducer to the repressor will remain repressed even in the presence of the inducer molecule, because the inducer cannot bind to the repressor on the operator locus in order to dere-press the operon. Similarly, bacteria harboring mutations in their lac operator locus such that the operator sequence will not bind a normal repressor molecule 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 (see below).

The Genetic Switch of Bacteriophage Lambda (A) Provides a Paradigm for Protein-DNA Interactions in Eukaryotic Cells

Like some eukaryotic viruses (eg, herpes simplex, HIV), some bacterial viruses can either reside in a dormant state within the host chromosomes or can replicate within the bacterium and eventually lead to lysis and killing of the bacterial host. Some E coli harbor such a "temperate" virus, bacteriophage lambda (X). When lambda infects an organism of that species it injects its 45,000-bp, double-stranded, linear DNA genome into the cell (Figure 39-4). Depending upon the nutritional state of the cell, the lambda DNA will either integrate into the host genome (lysogenic pathway) and remain dormant until activated (see below), or it will commence replicating until it has made about 100 copies of complete, protein-packaged virus, at which point it causes lysis of its host (lytic pathway). The newly generated virus particles can then infect other susceptible hosts.

When integrated into the host genome in its dormant state, lambda will remain in that state until activated by exposure of its lysogenic bacterial host to DNA-damaging agents. In response to such a noxious stimulus, the dormant bacteriophage becomes "induced" and begins to transcribe and subsequently translate those genes of its own genome which are necessary for its excision from the host chromosome, its DNA replication, and the synthesis of its protein coat and lysis enzymes. This event acts like a trigger or type C (Figure 39-1) response; ie, once lambda has committed itself to induction, there is no turning back until the cell is lysed and the replicated bacteriophage released. This switch from a dormant or prophage state to a lytic infection is well understood at the genetic and molecular levels and will be described in detail here.

The switching event in lambda is centered around an 80-bp region in its double-stranded DNA genome referred to as the "right operator" (Or) (Figure 39-5A). The right operator is flanked on its left side by the structural gene for the lambda repressor protein, the cI protein, and on its right side by the structural gene encoding another regulatory protein called Cro. When lambda is in its prophage state—ie, integrated into the host genome—the cl repressor gene is the only lambda gene cI protein that is expressed. When the bacterio-phage is undergoing lytic growth, the cl repressor gene is not expressed, but the cro gene—as well as many other genes in lambda—is expressed. That is, when the repressor gene is on, the cro gene is off, and when the cro gene is on, the repressor gene is off. As we shall see, these two genes regulate each other's expression and thus, ultimately, the decision between lytic and lysogenic growth of lambda. This decision between repressor gene transcription and cro gene transcription is a paradigmatic example of a molecular switch.

The 80-bp X right operator, Or, can be subdivided into three discrete, evenly spaced, 17-bp cis-active DNA elements that represent the binding sites for either of two bacteriophage X regulatory proteins. Impor-

pathway pathway pathway pathway

Figure 39-4. Infection of the bacterium Ecoli by phage lambda begins when a virus particle attaches itself to the bacterial cell (1) and injects its DNA (shaded line) into the cell (2, 3). Infection can take either of two courses depending on which of two sets of viral genes is turned on. In the lysogenic pathway, the viral DNA becomes integrated into the bacterial chromosome (4, 5), where it replicates passively as the bacterial cell divides. The dormant virus is called a prophage, and the cell that harbors it is called a lysogen. In the alternative lytic mode of infection, the viral DNA replicates itself (6) and directs the synthesis of viral proteins (7). About 100 new virus particles are formed. The proliferating viruses lyse, or burst, the cell (8). A prophage can be "induced" by a DNA damaging agent such as ultraviolet radiation (9). The inducing agent throws a switch, so that a different set of genes is turned on. Viral DNA loops out of the chromosome (10) and replicates; the virus proceeds along the lytic pathway. (Reproduced, with permission, from Ptashne M, Johnson AD, Pabo CO: A genetic switch in a bacterial virus. Sci Am [Nov] 1982;247:128.)

Gene for repressor (cl)

Gene for Cro

Repressor RNA

□ I 1111 1111 1111 1111 1111 1111 1111 1111 1111 11111 1111 1111 1111 1111 1111 1111 1111 1111 1111 1111 1111 1111 1111 1111 1111 I

Repressor promoter

--

cro Promoter

\

T

A

C C T C

T

G

G

C

G

G

T

G

A 1

T A

A

T

G G A G

A

C

C

G

C

C

A

C

T

A T

Figure 39-5. Right operator (OR) is shown in increasing detail in this series of drawings. The operator is a region of the viral DNA some 80 base pairs long (A). To its left lies the gene encoding lambda repressor (cI), to its right the gene (cro) encoding the regulator protein Cro. When the operator region is enlarged (B), it is seen to include three subregions, OR1, Or2, and OR3, each 17 base pairs long. They are recognition sites to which both repressor and Cro can bind. The recognition sites overlap two promoters—sequences of bases to which RNA polymerase binds in order to transcribe these genes into mRNA (wavy lines), that are translated into protein. Site OR1 is enlarged (C) to show its base sequence. Note that in this region of the X chromosome, both strands of DNA act as a template for transcription (Chapter 39). (Reproduced, with permission, from Ptashne M, Johnson AD, Pabo CO: A genetic switch in a bacterial virus. Sci Am [Nov] 1982;247:128.)

tantly, the nucleotide sequences of these three tandemly arranged sites are similar but not identical (Figure 39—5B). The three related cis elements, termed operators OR1, OR2, and OR3, can be bound by either cI or Cro proteins. However, the relative affinities of cI and Cro for each of the sites varies, and this differential binding affinity is central to the appropriate operation of the X phage lytic or lysogenic "molecular switch." The DNA region between the cro and repressor genes also contains two promoter sequences that direct the binding of RNA polymerase in a specified orientation, where it commences transcribing adjacent genes. One promoter directs RNA polymerase to transcribe in the rightward direction and, thus, to transcribe cro and other distal genes, while the other promoter directs the transcription of the repressor gene in the leftward direction (Figure 39-5B).

The product of the repressor gene, the 236-amino-acid, 27 kDa repressor protein, exists as a two-domain molecule in which the amino terminal domain binds to operator DNA and the carboxyl terminal domain promotes the association of one repressor protein with another to form a dimer. A dimer of re-pressor molecules binds to operator DNA much more tightly than does the monomeric form (Figure 39-6A to 39-6C).

The product of the cro gene, the 66-amino-acid, 9 kDa Cro protein, has a single domain but also binds the operator DNA more tightly as a dimer (Figure

39-6D). The Cro protein's single domain mediates both operator binding and dimerization.

In a lysogenic bacterium—ie, a bacterium containing a lambda prophage—the lambda repressor dimer binds preferentially to OR1 but in so doing, by a cooperative interaction, enhances the binding (by a factor of 10) of another repressor dimer to OR2 (Figure 39-7). The affinity of repressor for OR3 is the least of the three operator subregions. The binding of repressor to OR1 has two major effects. The occupation of OR1 by repressor blocks the binding of RNA polymerase to the right-ward promoter and in that way prevents expression of cro. Second, as mentioned above, repressor dimer bound to OR1 enhances the binding of repressor dimer to OR2. The binding of repressor to OR2 has the important added effect of enhancing the binding of RNA polym-erase to the leftward promoter that overlaps OR2 and thereby enhances transcription and subsequent expression of the repressor gene. This enhancement of transcription is apparently mediated through direct proteinprotein interactions between promoter-bound RNA polymerase and OR2-bound repressor. Thus, the lambda repressor is both a negative regulator, by preventing transcription of cro, and a positive regulator, by enhancing transcription of its own gene, the repressor gene. This dual effect of repressor is responsible for the stable state of the dormant lambda bacteriophage; not only does the repressor prevent expression of the genes necessary for lysis, but it also promotes expression of itself to

Amino acids 132 - 236

Amino acids 1 - 92

Amino acids 132 - 236

Amino acids 1 - 92

Cro

Figure 39-6. Schematic molecular structures of cI (lambda repressor, shown in A, B, and C) and Cro (D). Lambda repressor protein is a polypeptide chain 236 amino acids long. The chain folds itself into a dumbbell shape with two substructures: an amino terminal (NH2) domain and a carboxyl terminal (COOH) domain. The two domains are linked by a region of the chain that is susceptible to cleavage by proteases (indicated by the two arrows in A). Single repressor molecules (monomers) tend to associate to form dimers (B); a dimer can dissociate to form monomers again. A dimer is held together mainly by contact between the carboxyl terminal domains (hatching). Repressor dimers bind to (and can dissociate from) the recognition sites in the operator region; they display the greatest affinity for site OR1 (C). It is the amino terminal domain of the repressor molecule that makes contact with the DNA (hatching). Cro (D) has a single domain with sites that promote dimerization and other sites that promote binding of dimers to operator, preferentially to OR3. (Reproduced, with permission, from Ptashne M, Johnson AD, Pabo CO: A genetic switch in a bacterial virus. Sci Am [Nov] 1982;247:128.)

stabilize this state of differentiation. In the event that intracellular repressor protein concentration becomes very high, this excess repressor will bind to OR3 and by so doing diminish transcription of the repressor gene from the leftward promoter until the repressor concentration drops and repressor dissociates itself from OR3.

With such a stable, repressive, cI-mediated, lyso-genic state, one might wonder how the lytic cycle could ever be entered. However, this process does occur quite efficiently. When a DNA-damaging signal, such as ultraviolet light, strikes the lysogenic host bacterium, fragments of single-stranded DNA are generated that activate a specific protease coded by a bacterial gene and referred to as recA (Figure 39-7). The activated recA protease hydrolyzes the portion of the repressor protein that connects the amino terminal and carboxyl terminal domains of that molecule (see Figure 39-6A). Such cleavage of the repressor domains causes the repressor dimers to dissociate, which in turn causes dissociation of the repressor molecules from OR2 and eventually from OR1. The effects of removal of repressor from OR1 and OR2 are predictable. RNA polym-erase immediately has access to the rightward promoter and commences transcribing the cro gene, and the enhancement effect of the repressor at OR2 on leftward transcription is lost (Figure 39-7).

The resulting newly synthesized Cro protein also binds to the operator region as a dimer, but its order of preference is opposite to that of repressor (Figure 39-7). That is, Cro binds most tightly to OR3, but there is no cooperative effect of Cro at OR3 on the binding of Cro to OR2. At increasingly higher concentrations of Cro, the protein will bind to OR2 and eventually to OR1.

Occupancy of OR3 by Cro immediately turns off transcription from the leftward promoter and in that way prevents any further expression of the repressor gene. The molecular switch is thus completely "thrown" in the lytic direction. The cro gene is now expressed, and the repressor gene is fully turned off. This event is irreversible, and the expression of other lambda genes begins as part of the lytic cycle. When Cro repressor concentration becomes quite high, it will eventually occupy OR1 and in so doing reduce the expression of its own gene, a process that is necessary in order to effect the final stages of the lytic cycle.

The three-dimensional structures of Cro and of the lambda repressor protein have been determined by x-ray crystallography, and models for their binding and effecting the above-described molecular and genetic events have been proposed and tested. Both bind to DNA using helix-turn-helix DNA binding domain motifs (see below).

Prophage

RNA polymerase or3 or2 or1 I-1 I-1 I-1

RNA polymerase or3 or2 or1 I-1 I-1 I-1

Induction (1)

RNA polymerase

RNA polymerase

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

Get My Free Ebook


Post a comment