Like some eukaryotic viruses (eg, herpes simplex virus and HIV), certain 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 (λ). When lambda infects an organism of that species, it injects its 48,490-bp, double-stranded, linear DNA genome into the cell (Figure 1). Depending on the nutritional state of the cell, the lambda DNA will either integrate into the host genome (lysogenic pathway) and remain dormant until activated (see following discussion), 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 host cells. Poor growth conditions favor lysogeny while good growth conditions promote the lytic path way of lambda growth.

Fig1. Alternate lytic and lysogenic lifestyles of bacteriophage lambda. Infection of the bacterium E. coli by phage lambda begins when a virus particle attaches itself to specific receptors on the bacterial cell surface (1) and injects its DNA (dark green line) into the cell (2), where the phage genome then circularizes (3). Infection can take either of two courses depending on which two sets of viral genes is turned on. In the lysogenic pathway, the viral DNA becomes integrated into the bacterial chromosome (red) (4, 5), where it is replicated passively as part of the bacterial DNA during E. coli cell division. This dormant, bacterial genome-integrated virus is called a prophage, and the cell that harbors is called a lysogen. In the alternative, lytic mode of infection, the viral DNA excises from the E. coli chromosome and replicates itself (6) in order to direct the synthesis of viral proteins; black lines (7). About 100 new virus particles (green hexagons) are formed. The proliferating viruses induce lysis of the cell (8). A prophage can be “induced” by a DNA damaging agent such as ultraviolet radiation (9). The inducing agent throws a switch (see text and Figure 38–5; the λ “molecular switch.”), so that a different set of viral genes is turned on. Viral DNA loops out and is excised from the E. coli chromosome (10) and replicates; the virus then proceeds along the lytic pathway.

When integrated into the host genome in its dormant state, lambda will remain in that state until activated by exposure of its 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 that 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 response; that is, once dormant 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; though less well understood at the molecular level, HIV and herpes viruses can behave similarly, transitioning from dormant to active states in infected humans.

The lytic/lysogenic genetic switching event in lambda is centered around an 80-bp region in its double-stranded DNA genome referred to as the “right operator” (O_R ) (Figure 2A) The right operator is flanked on its left side by the gene for the lambda repressor protein, cI, and on its right side by the gene encoding another regulatory protein, the cro gene. When lambda is in its prophage state—that is, integrated into the host genome—the cI repressor gene is the only lambda gene that is expressed. When the bacteriophage is undergoing lytic growth, the cI repressor gene is not expressed, but the cro gene—as well as many other lambda genes—is expressed. Thus, when the cI repressor gene is on, the cro gene is off, and when the crogene is on, thecI 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 transcriptional switch.

Fig2. Genetic organization of the lambda lifestyle “molecular switch.” Right operator (OR ) is shown in increasing detail in this series of drawings. The operator is a region of the viral DNA some 80-bp 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 termed operators: OR 1, OR 2, and OR 3, each 17-bp long. These three DNA elements are recognition sites to which both λ cI repressor and Cro proteins can bind. The recognition sites overlap two divergent 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 OR 1 is enlarged (C) to show its base sequence. (Reproduced with permission from Alan D. Iselin, artist.)

The 80-bp lambda 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 lambda regulatory proteins. Importantly, the nucleotide sequences of these three tandemly arranged sites are similar but not identical (Figure 38–5B). The three related cis elements, termed operators OR 1, OR 2, and OR 3, can be bound Lytic pathway 6 by either cI or cro proteins. However, the relative affinities of cI and cro for each of the sites vary, and this differential binding affinity is central to the appropriate operation of the lambda 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 cI repressor gene in the leftward direction (see Figure 2B).

The product of the cI repressor gene, the 236-amino-acid λ cI repressor protein is a two-domain molecule with amino terminal DNA-binding domain (DBD)and carboxyl-terminal dimerization domain. Association of one repressor protein with another forms a dimer. cI repressor dimers bind to opera tor DNA much more tightly than do monomers (Figure 3A to 3C).

Fig3. Schematic molecular structures of lambda regulatory proteins cI and Cro. (A)The lambda repressor protein is a 236-amino-acid polypeptide. 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 less structured and susceptible to cleavage by proteases (indicated by the two arrows. (B) Single repressor molecules (monomers) tend to reversibly associate to form dimers. A dimer is held together mainly by contact between the carboxyl-terminal domains (green hatching). (C) cI repressor dimers bind to (and can dissociate from) the recognition sites in the operator region; they display differential affinities for the three operator sites, OR 1 > OR 2 > OR 3. The DNA-binding from Alan D. Iselin, artist.) domains (DBD) of the repressor molecule that makes contact with DNA (blue hatching). (D) Cro is a single globular protein that contains both a DNA binding domain (blue hatching) and a cro-cro dimerization domain, which promotes binding of cro-cro dimers to target operator DNA. It is important that cro exhibits the highest affinity for OR 3, opposite the sequence binding preference of the cI protein. (Reproduced with permission from Alan D. Iselin, artist.)

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 3D). The cro protein’s single domain mediates both operator binding and dimerization.

In a lysogenic bacterium—that is, a bacterium containing an integrated, dormant lambda prophage—the lambda repressor dimer binds preferentially to OR 1 but in so doing, by a cooperative interaction, enhances the binding (by a factor of 10) of another repressor dimer to OR 2 (Figure 4). The affinity of repressor for OR 3 is the least of the three operator subregions. The binding of repressor to OR 1 has two major effects. First, occupancy of OR 1 by repressor blocks the binding of RNA polymerase to the rightward promoter and in that way prevents expression of cro. Second, as mentioned earlier, repressor dimer bound to OR 1 enhances the binding of repressor dimer to OR 2. The binding of repressor to OR 2 has the important added effect of enhancing the binding of RNA polymerase to the leftward promoter that overlaps OR 3 and thereby enhances transcription and subsequent expression of the repressor gene. This enhancement of transcription is mediated through direct protein–protein interactions between promoter-bound RNA polymerase and OR 2-bound repressor, much as described earlier for CAP protein and RNA polymerase on the lac operon. Thus, the λ cI protein is both a negative regulator, by preventing transcription of cro, and a positive regulator, by enhancing transcription of its own gene, cI. 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 stabilize this state of differentiation. In the event that intracellular repressor protein concentration becomes very high, the excess repressor will bind to OR 3 and by so doing diminish transcription of the repressor gene from the leftward promoter, by blocking RNAP binding to the cI promoter, until the repressor concentration drops and repressor dissociates from OR 3. Similar examples of repressor proteins also having the ability to activate transcription have been observed in eukaryotes.

Fig4. Configuration of the lytic/lysogenic switch is shown at four stages of the lambda phage “life” cycle. The lysogenic pathway (in which the virus remains dormant as a prophage) is selected when a repressor dimer binds to OR 1, thereby making it likely that OR 2 will be bound immediately by another dimer due to the cooperative nature of cI-OR DNA binding. In the prophage (top), the repressor dimers bound at OR 1 and OR 2 prevent RNA polymerase from binding to the rightward cro promoter and so block the synthesis of cro (negative control). Simultaneously these DNA-bound cI proteins enhance the binding of polymerase to the leftward promoter (positive control), with the result that the repressor gene is transcribed into RNA (initiation at cI gene transcription start site; TSS) and more repressor is synthesized, maintaining the lysogenic state. The prophage is induced (middle) when ultraviolet radiation activates the protease recA, which cleaves cI repressor monomers. Induction (1) The equilibrium of free monomers, free cI dimers, and bound dimers is thereby shifted by mass action, and cI dimers thus dissociate from the operator sites. RNA polymerase is no longer stimulated to bind to the leftward promoter, so that repressor is no longer synthesized. As induction proceeds, Induction (2) all the operator sites become vacant, thus polymerase can bind to the rightward promoter and cro is synthesized (cro TSS shown). During early lytic growth, a single cro dimer binds to OR 3 (light blue shaded circles), the site for which it has the highest affinity thereby occluding the cI promoter. Consequently, RNA polymerase cannot bind to the leftward promoter, but the rightward promoter remains accessible. Polymerase continues to bind there, transcribing cro and other early lytic genes. Lytic growth ensues (bottom). (Reproduced with permission from Alan D. Iselin, artist.)

With such a stable, repressive, cI-mediated, lysogenic 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 co-protease coded by a bacterial gene and referred to as recA (see Figure 4). 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 38–6A). Such cleavage of the repressor domains causes the repressor dimers to dis sociate, which in turn causes dissociation of the repressor molecules from OR 2 and eventually from OR 1. The effects of removal of repressor from OR 1 and OR 2 are predictable. RNA polymerase immediately has access to the rightward promoter and commences transcribing the cro gene, while simultaneously the enhancing effect of the repressor at OR 2 on leftward transcription is lost as well (see Figure 4).

The resulting newly synthesized cro protein also binds to the operator region as a dimer, but as noted earlier, its order of preference is opposite to that of repressor (see Figure4). That is, cro binds most tightly to OR 3, but there is no cooperative effect of cro at OR 3 on the binding of cro to OR 2. At increasingly higher concentrations of cro, the protein will bind to OR 2 and eventually to OR 1.

Importantly, occupancy of OR 3 by cro immediately turns off transcription from the leftward cI promoter and in that way prevents any further expression of the cI 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 OR 1 and in so doing reduce the expression of its own gene, a process that is necessary in order to drive transcription of the genes needed for the final stages of the lytic cycle.

The three-dimensional structures of cro and of the λ cI repressor protein have been determined by x-ray crystallography, and models for their binding and driving the above-described molecular and genetic events have been formulated and tested. Both bind DNA using helix-turn-helix DBD motifs (see following discussion). Along with regulation of the expression of the lac operon, the λ molecular switch described here provides arguably the best understanding of the molecular events involved in gene transcription activation and repression.

Detailed analysis of the λ repressor led to the important concept that transcription regulatory proteins have several functional domains. For example, lambda repressor binds to DNA with high affinity. Repressor monomers form dimers that cooperatively interact with each other, these proteins can interact with RNA polymerase, to enhance or block promoter binding or RNAP open complex formation. The protein-DNA interface and the three protein–protein interfaces all involve separate and distinct domains of the two molecules. As will be noted later, this is a characteristic that is typical of most molecules that regulate transcription.

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مواضيع ذات صلة

Epigenetic Mechanisms Contribute Importantly to the Control of Gene Transcription

The Chromatin Template Contributes Importantly to Eukaryotic Gene Transcription Control

Analysis of Lactose Metabolism in E. coli Led to the Discovery of the Basic Principles of Gene Transcription

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

Like Transcription, Protein synthesis can be described in three phases: Initiation, Elongation, & Termination

Mutations result when changes occur in the Nucleotide Sequence

The Genetic Code is Degenerate, Unambiguous, Nonoverlapping, Without Punctuation, & Universal

RNAs can be Extensively Modified

RNA Molecules are Extensively Processed Before They Become Functional

Phosphorylation Activates Pol II