Lewin’s Genes XII

25.1 Introduction

A virus consists of a nucleic acid genome contained in a protein coat. In order to reproduce, the virus must infect a host cell. The typical pattern of an infection is to subvert the functions of the host cell for the purpose of producing a large number of progeny viruses. Viruses that infect bacteria are generally called bacteriophages, often abbreviated as phages or simply ϕ. Usually, a phage infection kills the bacterium. The process by which a phage infects a bacterium, reproduces itself, and then kills its host is called lytic infection. In the typical lytic cycle, the phage DNA (or RNA) enters the host bacterium, its genes are transcribed in a set order, the phage genetic material is replicated, and the protein components of the phage particle are produced. Finally, the host bacterium is broken open (lysed) to release the assembled progeny particles by the process of lysis. For some phages, called virulent phages, this is their only strategy for survival.

Other phages have a dual existence. They are able to perpetuate themselves via the same sort of lytic cycle in what amounts to an open strategy for producing as many copies of the phage as rapidly as possible. They also have an alternative form of existence, though, in which the phage genome is present in the bacterial genome in a latent form known as a prophage. This form of propagation is called lysogeny, and the infected bacteria are known as lysogens. Phages that follow this pathway are called temperate phages.

In a lysogenic bacterium, the prophage is inserted, or recombined, into the bacterial genome and is inherited in the same way as bacterial genes. The process by which it is converted from an independent phage genome into a prophage that is a linear part of the bacterial genome is described as integration. By virtue of its possession of a prophage, a lysogenic bacterium has immunity against infection by other phage particles of the same type. Immunity is established by a single integrated prophage, so in general a bacterial genome contains only one copy of a prophage of any particular type.

Transitions occur between the lysogenic and lytic modes of existence. FIGURE 25.1 shows that when a temperate phage produced by a lytic cycle enters a new bacterial host cell it either repeats the lytic cycle or enters the lysogenic state. The outcome depends on the conditions of infection and the genotypes of the phage and the bacterium.

FIGURE 25.1 Lytic development involves the reproduction of phage particles with destruction of the host bacterium, but lysogenic existence allows the phage genome to be carried as part of the bacterial genetic information.

A prophage is freed from the restrictions of lysogeny by a process called induction. First, the phage DNA is released from the bacterial chromosome by another recombination event called excision; the free DNA then proceeds through the lytic pathway.

The alternative forms in which these phages are propagated are determined by the regulation of transcription. Lysogeny is maintained by the interaction of a phage repressor with an operator. The lytic cycle requires a cascade of transcriptional controls. The transition between the two lifestyles is accomplished by the establishment of repression (lytic cycle to lysogeny) or by the relief of repression (induction of lysogen to lytic phage). These regulatory processes provide a wonderful example of how a series of relatively simple regulatory actions can be built up into complex developmental pathways.

25.2 Lytic Development Is Divided into Two Periods

Phage genomes by necessity are small. As with all viruses, they are restricted by the need to package the nucleic acid within the protein coat. This limitation dictates many of the viral strategies for reproduction. Typically, a virus takes over the apparatus of the host cell, which then replicates and expresses phage genes instead of the bacterial genes.

Usually, the phage has genes whose function is to ensure preferential replication of phage DNA. These genes are concerned with the initiation of replication and may even include a new DNA polymerase. Changes are introduced in the capacity of the host cell to engage in transcription. They involve replacing the RNA polymerase or modifying its capacity for initiation or termination. The result is always the same: Phage mRNAs are preferentially transcribed. As far as protein synthesis is concerned, the phage is, for the most part, content to use the host apparatus, redirecting its activities principally by replacing bacterial mRNA with phage mRNA.

Lytic development is accomplished by a pathway in which the phage genes are expressed in a particular order. This ensures that the right amount of each component is present at the appropriate time. The cycle can be divided into the two general parts illustrated in FIGURE 25.2:

Early infection describes the period from entry of the DNA to the start of its replication.
Late infection defines the period from the start of replication to the final step of lysing the bacterial cell to release progeny phage particles.

FIGURE 25.2 Lytic development takes place by producing phage genomes and protein particles that are assembled into progeny phages.

The early phase is devoted to the production of enzymes involved in the reproduction of DNA. These include the enzymes concerned with DNA synthesis, recombination, and sometimes modification. Their activities cause a pool of phage genomes to accumulate. In this pool, genomes are continually replicating and recombining, so that the events of a single lytic cycle concern a population of phage genomes.

During the late phase, the protein components of the phage particle are synthesized. Often, many different proteins are needed to make up head and tail structures, so the largest part of the phage genome consists of late functions. In addition to the structural proteins, “assembly proteins” are needed to help construct the particle, although they are not incorporated into it themselves. By the time the structural components are assembling into heads and tails, replication of DNA has reached its maximum rate. The genomes then are inserted into the empty protein heads, tails are added, and the host cell is lysed to allow release of new viral particles.

25.3 Lytic Development Is Controlled by a Cascade

The organization of the phage genetic map often reflects the sequence of lytic development. The concept of the operon is taken to somewhat of an extreme, in which the genes coding for proteins with related functions are clustered to allow their control with the maximum economy. This allows the pathway of lytic development to be controlled with a small number of regulatory switches.

The lytic cycle is under positive control, so that each group of phage genes can be expressed only when an appropriate signal is given. FIGURE 25.3 shows that the regulatory genes function in a cascade, in which a gene expressed at one stage is necessary for synthesis of the genes that are expressed at the next stage.

FIGURE 25.3 Phage lytic development proceeds by a regulatory cascade, in which a gene product at each stage is needed for expression of the genes at the next stage.

The early part of the first stage of gene expression necessarily relies on the transcription apparatus of the host cell. In general, only a few genes are expressed at this time. Their promoters are indistinguishable from those of host genes. The name of this class of genes depends on the phage. In most cases, they are known as the early genes. In phage lambda, they are given the evocative description of immediate early genes. Irrespective of the name, they constitute only a preliminary set of genes, representing just the initial part of the early period. Sometimes they are exclusively occupied with the transition to the next period. In all cases, one of these genes always encodes a protein, a gene regulator that is necessary for transcription of the next class of genes.

This next class of genes in the early stage is known variously as the delayed early or middle gene group. Its expression typically starts as soon as the regulator protein coded by the early gene(s) is available. Depending on the nature of the control circuit, the initial set of early genes may or may not continue to be expressed at this stage. If control is at transcription initiation, the two events are independent (as shown in FIGURE 25.4), and early genes can be switched off when middle genes are transcribed. If control is at transcription termination, the early genes must continue to be expressed, as shown in FIGURE 25.5. Often, the expression of host genes is reduced. Together the two sets of early genes account for all necessary phage functions except those needed to assemble the particle coat itself and to lyse the cell.

FIGURE 25.4 Control at initiation utilizes independent transcription units, each with its own promoter and terminator, which produce independent mRNAs. The transcription units need not be located near one another.

FIGURE 25.5 Control at termination requires adjacent units so that transcription can read from the first gene into the next gene. This produces a single mRNA that contains both sets of genes.

When the replication of phage DNA begins, it is time for the late genes to be expressed. Their transcription at this stage usually is arranged by embedding an additional regulator gene within the previous (delayed early or middle) set of genes. This regulator may be another antitermination factor (as in lambda) or it may be another sigma factor (such as the Bacillus subtilis factor).

A lytic infection often falls into the stages just described, beginning with the early genes transcribed by host RNA polymerase (sometimes the regulators are the only products at this stage). This stage is followed by those genes transcribed under the direction of the regulator produced in the first stage (most of these genes encode enzymes needed for replication of phage DNA). The final stage consists of genes for phage components, which are transcribed under the direction of a regulator synthesized in the second stage.

The use of these successive controls, in which each set of genes contains a regulator that is necessary for expression of the next set, creates a cascade in which groups of genes are turned on (and sometimes off) at particular times. The means used to construct each phage cascade are different, but the results are similar.

25.4 Two Types of Regulatory Events Control the Lytic Cascade

At every stage of phage expression, one or more of the active genes is a regulator that is needed for the subsequent stage. The regulator may take the form of a new sigma factor that redirects the specificity of the host RNA polymerase or an antitermination factor that allows it to read a new group of genes (see the Prokaryotic Transcription chapter). The following discussion compares the use of switching at initiation or termination to control gene expression.

One mechanism for recognizing new phage promoters is to replace the sigma factor of the host enzyme with another factor that redirects its specificity in initiation, as shown in FIGURE 25.6. An alternative is to synthesize a new phage RNA polymerase. In either case, the critical feature that distinguishes the new set of genes is their possession of different promoters from those originally recognized by host RNA polymerase. Figure 25.4 shows that the two sets of transcripts are independent; as a consequence, early gene expression can cease after the new sigma factor or polymerase has been produced.

FIGURE 25.6 A phage may control transcription at initiation either by synthesizing a new sigma factor that replaces the host sigma factor or by synthesizing a new RNA polymerase.

Antitermination provides an alternative mechanism for phages to control the switch from early genes to the next stage of expression. The use of antitermination depends on a particular arrangement of genes. Figure 25.5 shows that the early genes lie adjacent to the genes that are to be expressed next, but are separated from them by terminator sites. If termination is prevented at these sites, the polymerase reads through into the genes on the other side. So in antitermination, the same promoters continue to be recognized by RNA polymerase. The new genes are expressed only by extending the RNA chain to form molecules that contain the early gene sequences at the 5′ end and the new gene sequences at the 3′ end. The two types of sequences remain linked; thus, early gene expression inevitably continues.

The regulator gene that controls the switch from immediate early to delayed early expression in phage lambda is identified by mutations in gene N that can transcribe only the immediate early genes; they proceed no further into the infective cycle (see Figure 25.10, later in this chapter). From the genetic point of view, the mechanisms of new initiation and antitermination are similar. Both are positive controls in which an early gene product must be made by the phage in order to express the next set of genes. By employing either sigma factor or antitermination proteins with different specifications, a cascade for gene expression can be constructed.

25.5 The Phage T7 and T4 Genomes Show Functional Clustering

The genome of phage T7 has three classes of genes, each of which constitutes a group of adjacent loci. As FIGURE 25.7 shows, the class I genes are the immediate early type and are expressed by host RNA polymerase as soon as the phage DNA enters the cell. Among the products of these genes are a phage RNA polymerase and enzymes that interfere with host gene expression. The phage RNA polymerase is responsible for expressing the class II genes (which are concerned principally with DNA synthesis functions) and the class III genes (which are concerned with assembling the mature phage particle).

FIGURE 25.7 Phage T7 contains three classes of genes that are expressed sequentially. The genome is ~38 kb.

Phage T4 has one of the larger phage genomes (165 kb), which is organized with extensive functional grouping of genes. FIGURE 25.8 presents the genetic map. Essential genes are numbered: A mutation in any one of these loci prevents successful completion of the lytic cycle. Nonessential genes are indicated by three-letter abbreviations. (They are defined as nonessential under the usual conditions of infection. We do not really understand the inclusion of many nonessential genes, but presumably they confer a selective advantage in some of T4’s habitats. In smaller phage genomes, most or all of the genes are essential.)

FIGURE 25.8 The map of T4 is circular. T4 has extensive clustering of genes encoding components of the phage and processes such as DNA replication, but there is also dispersion of genes encoding a variety of enzymatic and other functions. Essential genes are indicated by numbers. Nonessential genes are identified by letters. Only some representative T4 genes are shown on the map.

Three phases of gene expression have been identified. A summary of the functions of the genes expressed at each stage is shown in FIGURE 25.9. The early genes are transcribed by host RNA polymerase. The middle genes are also transcribed by host RNA polymerase, but two phage-encoded products, MotA and AsiA, also are required. The middle promoters lack a consensus –35 sequence and instead have a binding sequence for MotA. The phage protein is an activator that compensates for the deficiency in the promoter by assisting host RNA polymerase to bind. (This is similar to a mechanism employed by phage lambda with its cII gene, which is illustrated later in Figure 25.30 in the section The cII and cIII Genes Are Needed to Establish Lysogeny.) The early and middle genes account for virtually all of the phage functions concerned with the synthesis of DNA, modifying cell structure, and transcribing and translating phage genes.

The two essential genes in the “transcription” category fulfill a regulatory function: Their products are necessary for late gene expression. Phage T4 infection depends on a mechanical link between replication and late gene expression. Only actively replicating DNA can be used as a template for late gene transcription. The connection is generated by introducing a new sigma factor and also by making other modifications in the host RNA polymerase so that it is active only with a template of replicating DNA. This link establishes a correlation between the synthesis of phage protein components and the number of genomes available for packaging.

FIGURE 25.9 The phage T4 lytic cascade falls into two parts: Early functions are concerned with DNA synthesis; late functions with particle assembly.

25.6 Lambda Immediate Early and Delayed Early Genes Are Needed for Both Lysogeny and the Lytic Cycle

One of the most intricate cascade circuits is provided by phage lambda. Actually, the cascade for lytic development itself is straightforward, with two regulators controlling the successive stages of development. The circuit for the lytic cycle, though, is interlocked with the circuit for establishing lysogeny, as illustrated in FIGURE 25.10.

FIGURE 25.10 The lambda lytic cascade is interlocked with the circuitry for lysogeny.

When lambda DNA enters a new host cell, the lytic and lysogenic pathways start off the same way. Both require expression of the immediate early and delayed early genes, but then they diverge: Lytic development follows if the late genes are expressed, and lysogeny ensues if synthesis of a gene regulator called the lambda repressor is established by turning on its gene, the cI gene. Lambda has only two immediate early genes, transcribed independently by host RNA polymerase:

The N gene encodes an antitermination factor whose action at nut (N utilization) sites allows transcription to proceed into the delayed early genes (see the Prokaryotic Transcription chapter). The N gene is required for both the lytic and lysogenic pathways.
The cro gene encodes a repressor that prevents expression of the c1 gene encoding the lambda repressor (essentially derepressing the late genes, a necessary action if the lytic cycle is to proceed). It also turns off expression of the immediate early genes (which are not needed later in the lytic cycle). The lambda repressor is the major regulator required for lysogenic development.

The delayed early genes, turned on by the product of the N gene, include two replication genes (needed for lytic infection), seven recombination genes (some involved in recombination during lytic infection, two genes necessary to integrate lambda DNA into the bacterial chromosome for lysogeny), and three regulator genes. These regulator genes have opposing functions:

The cII–cIII pair of regulator genes is needed to establish the synthesis of the lambda repressor for the lysogenic pathway.
The Q regulator gene codes for an antitermination factor that allows host RNA polymerase to transcribe the late genes and is necessary for the lytic cycle.

Thus, the delayed early genes serve two masters: Some are needed for the phage to enter lysogeny, and the others are concerned with controlling the order of the lytic cycle. At this point, lambda is keeping open the option to choose either pathway.

25.7 The Lytic Cycle Depends on Antitermination by pN

To disentangle the lytic and lysogenic pathways, let’s first consider just the lytic cycle. FIGURE 25.11 gives the map of lambda phage DNA. A group of genes concerned with regulation is surrounded by genes needed for recombination and replication. The genes coding for structural components of the phage are clustered. All of the genes necessary for the lytic cycle are expressed in polycistronic transcripts from three promoters.

FIGURE 25.11 The lambda map shows clustering of related functions. The genome is 48,514 bp.

FIGURE 25.12 shows that the two immediate early genes, N and cro, are transcribed by host RNA polymerase. N is transcribed toward the left and cro toward the right. Each transcript is terminated at the end of the gene. The protein pN is the regulator, the antitermination factor that allows transcription to continue into the delayed early genes by suppressing use of the terminators t_L and t_R (see the Prokaryotic Transcription chapter). In the presence of pN, transcription continues to the left of the N gene into the recombination genes and to the right of the cro gene into the replication genes.

FIGURE 25.12 Phage lambda has two early transcription units. In the “leftward” unit, the “upper” strand is transcribed toward the left; in the “rightward” unit, the “lower” strand is transcribed toward the right. Genes N and cro are the immediate early functions and are separated from the delayed early genes by the terminators. Synthesis of N protein allows RNA polymerase to pass the terminators t_L1 to the left and t_R1 to the right.

The map in Figure 25.11 gives the organization of the lambda DNA as it exists in the phage particle. Shortly after infection, though, the ends of the DNA join to form a circle. FIGURE 25.13 shows the true state of lambda DNA during infection. The late genes are welded into a single group, which contains the lysis genes S–R from the right end of the linear DNA and the head and tail genes A–J from the left end.

FIGURE 25.13 Lambda DNA circularizes during infection, so that the late gene cluster is intact in one transcription unit.

The late genes are expressed as a single transcription unit, starting from a promoter P_R′ that lies between Q and S. The late promoter is used constitutively. In the absence of the product of gene Q (which is the last gene in the rightward delayed early unit), however, late transcription terminates at a site t_R3. The transcript resulting from this termination event is 194 bases long; it is known as 6S RNA. When pQ becomes available, it suppresses termination at t_R3 and the 6S RNA is extended, with the result that the late genes are expressed.

25.8 Lysogeny Is Maintained by the Lambda Repressor Protein

Looking at the lambda lytic cascade, we see that the entire program is set in motion by the initiation of transcription at the two promoters P_L and P_R for the immediate early genes N and cro. Lambda uses antitermination to proceed to the next stage of (delayed early) expression; therefore, the same two promoters continue to be used throughout the early period.

The expanded map of the regulatory region drawn in FIGURE 25.14 shows that the promoters P_L and P_R lie on either side of the cI gene. Associated with each promoter is an operator (O_L, O_R) at which repressor protein binds to prevent RNA polymerase from initiating transcription. The sequence of each operator overlaps with the promoter that it controls, and because this occurs so often these sequences are described as the P_L/O_L and P_R/O_R control regions.

FIGURE 25.14 The lambda regulatory region contains a cluster of trans-acting functions and cis-acting elements.

As a result of the sequential nature of the lytic cascade, the control regions provide a pressure point at which entry to the entire cycle can be controlled. By denying RNA polymerase access to these promoters, the lambda repressor protein prevents the phage genome from entering the lytic cycle. The lambda repressor functions in the same way as repressors of bacterial operons: It binds to specific operators.

The lambda repressor protein is encoded by the cI gene. Note in Figure 25.14 that the cI gene has two promoters, P_RM (promoter right maintenance) and P_RE (promoter right establishment). Mutants in this gene cannot maintain lysogeny but always enter the lytic cycle. In the time since the original isolation of the lambda repressor protein, the characterization of the repressor protein has shown how it both maintains the lysogenic state and provides immunity for a lysogen against superinfection by new phage lambda genomes.

The lambda repressor binds independently to the two operators, O_L and O_R. Its ability to repress transcription at the associated promoters is illustrated in FIGURE 25.15.

FIGURE 25.15 Repressor acts at the left operator and right operator to prevent transcription of the immediate early genes (N and cro). It also acts at the promoter P_RM to activate transcription by RNA polymerase of its own gene.

At O_L, the lambda repressor has the same sort of effect as has already been discussed for several other systems: It prevents RNA polymerase from initiating transcription at P_L. This stops the expression of gene N. P_L is used for all leftward early gene transcription; thus, this action prevents expression of the entire leftward early transcription unit, blocking the lytic cycle before it can proceed beyond early stages.

At O_R, repressor binding prevents the use of P_R, and so cro and the other rightward early genes cannot be expressed. The lambda repressor protein binding at O_R also stimulates transcription of cI, its own gene from P_RM.

The nature of this control circuit explains the biological features of lysogenic existence. Lysogeny is stable because the control circuit ensures that, so long as the level of lambda repressor is adequate, expression of the cI gene continues. The result is that O_L and O_R remain occupied indefinitely. By repressing the entire lytic cascade, this action maintains the prophage in its inert form.

25.9 The Lambda Repressor and Its Operators Define the Immunity Region

The presence of lambda repressor explains the phenomenon of immunity. If a second lambda phage DNA enters a lysogenic cell, repressor protein synthesized from the resident prophage genome will immediately bind to O_L and O_R in the new genome. This prevents the second phage from entering the lytic cycle.

The operators were originally identified as the targets for repressor action by virulent mutations (λvir). These mutations prevent the repressor from binding at O_L or O_R, with the result that the phage inevitably proceeds into the lytic pathway when it infects a new host bacterium. Note that λvir mutants can grow on lysogens because the virulent mutations in O_L and O_R allow the incoming phage to ignore the resident repressor and thus enter the lytic cycle. Virulent mutations in phages are the equivalent of operator-constitutive mutations in bacterial operons.

A prophage is induced to enter the lytic cycle when the lysogenic circuit is broken. This happens when the repressor is inactivated (see the next section, The DNA-Binding Form of the Lambda Repressor Is a Dimer). The absence of repressor allows RNA polymerase to bind at P_L and P_R, starting the lytic cycle, as shown in FIGURE 25.16.

FIGURE 25.16 In the absence of repressor, RNA polymerase initiates at the left and right promoters. It cannot initiate at P_RM in the absence of repressor.

The autoregulatory nature of the repressor maintenance circuit creates a sensitive response. The presence of the lambda repressor is necessary for its own synthesis; therefore, expression of the cI gene stops as soon as the existing repressor is destroyed. Thus, no repressor is synthesized to replace the molecules that have been damaged. This enables the lytic cycle to start without interference from the circuit that maintains lysogeny.

The region including the left and right operators, the cI gene, and the cro gene determines the immunity of the phage. Any phage that possesses this region has the same type of immunity, because it specifies both the repressor protein and the sites on which the repressor acts. Accordingly, this is called the immunity region (as marked in Figure 25.14). Each of the four lambdoid phages ϕ80, 21, 434, and λ has a unique immunity region. When we say that a lysogenic phage confers immunity to any other phage of the same type, we mean more precisely that the immunity is to any other phage that has the same immunity region (irrespective of differences in other regions).

25.10 The DNA-Binding Form of the Lambda Repressor Is a Dimer

The lambda repressor subunit is a polypeptide of 27 kD with the two distinct domains shown in FIGURE 25.17:

The N-terminal domain, residues 1–92, provides the operator-binding site.
The C-terminal domain, residues 132–236, is responsible for dimerization.

FIGURE 25.17 The N-terminal and C-terminal regions of repressor form separate domains. The C-terminal domains associate to form dimers; the N-terminal domains bind DNA.

The two domains are joined by a connector of 40 residues. When repressor is digested by a protease, each domain is released as a separate fragment.

Each domain can exercise its function independently of the other. The C-terminal fragment can form oligomers. The N-terminal fragment can bind the operators, though with a lower affinity than the intact lambda repressor. Thus, the information for specifically contacting DNA is contained within the N-terminal domain, but the efficiency of the process is enhanced by the attachment of the C-terminal domain.

The dimeric structure of the lambda repressor is crucial in maintaining lysogeny. The induction of a lysogenic prophage into the lytic cycle is caused by cleavage of the repressor subunit in the connector region, between residues 111 and 113. (This is a counterpart to the allosteric change in conformation that results when a small-molecule inducer inactivates the repressor of a bacterial operon, a capacity that the lysogenic repressor does not have.) Induction occurs under certain adverse conditions, such as exposure of lysogenic bacteria to ultraviolet (UV) irradiation, which leads to proteolytic inactivation of the repressor due to the induction of the SOS damage response system.

In the intact state, dimerization of the C-terminal domains ensures that when the repressor binds to DNA, its two N-terminal domains each contact DNA simultaneously. Cleavage releases the C-terminal domains from the N-terminal domains, though. As illustrated in FIGURE 25.18, this means that the N-terminal domains can no longer dimerize, which upsets the equilibrium between monomers and dimers. As a result, they do not have sufficient affinity for the lambda repressor to remain bound to DNA, which allows the lytic cycle to start. Also, two dimers usually cooperate to bind at an operator, and the cleavage destabilizes this interaction.

FIGURE 25.18 Repressor dimers bind to the operator. The affinity of the N-terminal domains for DNA is controlled by the dimerization of the C-terminal domains.

The balance between lysogeny and the lytic cycle depends on the concentration of repressor. Intact repressor is present in a lysogenic cell at a concentration sufficient to ensure that the operators are occupied. If the repressor is cleaved, however, this concentration is inadequate, because of the lower affinity of the separate N-terminal domain for the operator. A concentration of repressor that is too high would make it impossible to induce the lytic cycle in this way; a level that is too low, of course, would make it impossible to maintain lysogeny.

25.11 The Lambda Repressor Uses a Helix-Turn-Helix Motif to Bind DNA

A repressor dimer is the unit that binds to DNA. It recognizes a sequence of 17 bp displaying partial symmetry about an axis through the central base pair. FIGURE 25.19 shows an example of a binding site. The sequence on each side of the central base pair is sometimes called a half-site. Each individual N-terminal region contacts a half-site. Several DNA-binding proteins that regulate bacterial transcription share a similar mode of holding DNA, in which the active domain contains two short regions of α-helix that contact DNA. (Some transcription factors in eukaryotic cells use a similar motif; see the Eukaryotic Transcription Regulation chapter.)

FIGURE 25.19 The operator is a 17-bp sequence with an axis of symmetry through the central base pair. Each half-site is marked in light blue. Base pairs that are identical in each operator half are in dark blue.

The N-terminal domain of lambda repressor contains several stretches of α-helix, which are arranged as illustrated diagrammatically in FIGURE 25.20. Two of the helical regions are responsible for binding DNA. The helix-turn-helix model for contact is illustrated in FIGURE 25.21. Looking at a single monomer, α-helix-3 consists of nine amino acids, each of which lies at an angle to the preceding region of seven amino acids that forms α-helix-2. In the dimer, the two apposed helix-3 regions lie 34 Å apart, enabling them to fit into successive major grooves of DNA. The helix-2 regions lie at an angle that would place them across the groove. The symmetrical binding of dimer to the site means that each N-terminal domain of the dimer contacts a similar set of bases in its half-site.

FIGURE 25.20 Lambda repressor’s N-terminal domain contains five stretches of α-helix; helices 2 and 3 bind DNA.

FIGURE 25.21 In the two-helix model for DNA binding, helix-3 of each monomer lies in the wide groove on the same face of DNA and helix-2 lies across the groove.

Related forms of the α-helical motifs employed in the helix-turn-helix of the lambda repressor are found in several DNA-binding proteins, including catabolite repressor protein (CRP), the lac repressor, and several other phage repressors. By comparing the abilities of these proteins to bind DNA, the roles of each helix can be defined:

Contacts between helix-2 and helix-3 are maintained by interactions between hydrophobic amino acids.
Contacts between helix-3 and DNA rely on hydrogen bonds between the amino acid side chains and the exposed positions of the base pairs. This helix is responsible for recognizing the specific target DNA sequence and is therefore also known as the recognition helix. Comparison of the contact patterns illustrated in FIGURE 25.22 shows that the lambda repressor and Cro select different sequences in the DNA as their most favored targets because they have different amino acids in the corresponding positions in helix-3.
Contacts from helix-2 to the DNA take the form of hydrogen bonds connecting with the phosphate backbone. These interactions are necessary for binding, but do not control the specificity of target recognition. In addition to these contacts, a large part of the overall energy of interaction with DNA is provided by ionic interactions with the phosphate backbone.

FIGURE 25.22 Two proteins that use the two-helix arrangement to contact DNA recognize lambda operators with affinities determined by the amino acid sequence of helix-3.

What happens if we manipulate the coding sequence to construct a new protein by substituting the recognition helix in one repressor with the corresponding sequence from a closely related repressor? The specificity of the hybrid protein is that of its new recognition helix. The amino acid sequence of this short region determines the sequence specificities of the individual proteins and is able to act in conjunction with the rest of the polypeptide chain.

The bases contacted by helix-3 lie on one face of the DNA, as can be seen from the positions indicated on the helical diagram in Figure 25.22. Repressor makes an additional contact with the other face of DNA, though. The last six N-terminal amino acids of the N-terminal domain form an “arm” extending around the back. FIGURE 25.23 shows the view from the back. Lysine residues in the arm make contact with G residues in the major groove, and also with the phosphate backbone. The interaction between the arm and DNA contributes heavily to DNA binding; the binding affinity of a mutant armless repressor is reduced by about 1,000-fold.

FIGURE 25.23 A view from the back shows that the bulk of the repressor contacts one face of DNA, but its N-terminal arms reach around to the other face.

25.12 Lambda Repressor Dimers Bind Cooperatively to the Operator

Each operator contains three repressor-binding sites. As can be seen in FIGURE 25.24, no two of the six individual repressor-binding sites are identical, but they all conform to a consensus sequence. The binding sites within each operator are separated by spacers of 3 to 7 bp that are rich in A-T base pairs. The sites at each operator are numbered so that O_R consists of the series of binding sites O_R1-O_R2-O_R3, whereas O_L consists of the series O_L1-O_L2-O_L3. In each case, site 1 lies closest to the start point for transcription in the promoter, and sites 2 and 3 lie farther upstream.

FIGURE 25.24 Each operator contains three repressor-binding sites and overlaps with the promoter at which RNA polymerase binds. The orientation of O_L has been reversed from usual to facilitate comparison with O_R.

Faced with the triplication of binding sites at each operator, how does the lambda repressor decide where to start binding? At each operator, site 1 has a greater affinity (roughly 10-fold) than the other sites for the lambda repressor. Thus, it always binds first to O_L1 and O_R1.

Lambda repressor binds to subsequent sites within each operator in a cooperative manner. The presence of a dimer at site 1 greatly increases the affinity with which a second dimer can bind to site 2. When both sites 1 and 2 are occupied, this interaction does not extend farther, to site 3. At the concentrations of the lambda repressor usually found in a lysogen, both sites 1 and 2 are filled at each operator, but site 3 is not occupied.

The C-terminal domain is responsible for the cooperative interaction between dimers, as well as for the dimer formation between subunits. FIGURE 25.25 shows that it involves both subunits of each dimer; that is, each subunit contacts its counterpart in the other dimer, forming a tetrameric structure.

FIGURE 25.25 When two lambda repressor dimers bind cooperatively, each of the subunits of one dimer contacts a subunit in the other dimer.

A result of cooperative binding is the increase in effective affinity of repressor for the operator at physiological concentrations. This enables a lower concentration of repressor to achieve occupancy of the operator. This is an important consideration in a system in which release of repression has irreversible consequences. In an operon coding for metabolic enzymes, after all, failure to repress will merely allow unnecessary synthesis of enzymes. Failure to repress lambda prophage, however, will lead to induction of phage and lysis of the cell.

The sequences shown in Figure 25.22 indicate that O_L1 and O_R1 lie more or less in the center of the RNA polymerase binding sites of P_L and P_R, respectively. Occupancy of O_L1-O_L2 and O_R1-O_R2 thus physically blocks access of RNA polymerase to the corresponding promoters.

25.13 The Lambda Repressor Maintains an Autoregulatory Circuit

Once lysogeny has been established, the cI gene is transcribed from the P_RM promoter (see Figure 25.14) that lies to its right, close to P_R/O_R. Transcription terminates at the left end of the gene. The mRNA starts with the AUG initiation codon; because of the absence of a 5′ untranslated region (UTR) containing a ribosome-binding site, this is a very poor message that is translated inefficiently, producing only a low level of protein. Establishment of transcription for the cI gene is described later in this chapter in the section The Cro Repressor Is Needed for Lytic Infection.

The presence of the lambda repressor at O_R has dual effects, as noted earlier in the section Lysogeny Is Maintained by the Lambda Repressor Protein. It blocks expression from P_R, but it assists transcription from P_RM. RNA polymerase can initiate efficiently at P_RM only when the lambda repressor is bound at O_R. The lambda repressor thus behaves as a positive regulator protein that is necessary for transcription of its own gene, cI. This is the definition of an autoregulatory circuit.

At O_L, the repressor has the same sort of effect. It prevents RNA polymerase from initiating transcription at P_L; this stops the expression of gene N. P_L is used for all leftward early gene transcription. As a result, this action prevents expression of the entire leftward early transcription unit. Thus, the lytic cycle is blocked before it can proceed beyond early stages. Its actions at O_R and O_L are summarized in FIGURE 25.26.

FIGURE 25.26 Positive control mutations identify a small region at helix-2 that interacts directly with RNA polymerase.

The RNA polymerase binding site at P_RM is adjacent to O_R2. This explains how the lambda repressor autoregulates its own synthesis. When two dimers are bound at O_R1-O_R2, the amino terminal domain of the dimer at O_R2 interacts with RNA polymerase. The nature of the interaction is identified by mutations in the repressor that abolish positive control because they cannot stimulate RNA polymerase to transcribe from P_RM. They map within a small group of amino acids, located on the outside of helix-2 or in the turn between helix-2 and helix-3. The mutations reduce the negative charge of the region; conversely, mutations that increase the negative charge enhance the activation of RNA polymerase. This suggests that the group of amino acids constitutes an “acidic patch” that functions by an electrostatic interaction with a basic region on RNA polymerase to activate it.

The location of these “positive control mutations” in the repressor is indicated in FIGURE 25.27. They lie at a site on repressor that is close to a phosphate group on DNA, which is also close to RNA polymerase. Thus, the group of amino acids on repressor that is involved in positive control is in a position to contact the polymerase. The important principle is that protein–protein interactions can release energy that is used to help to initiate transcription.

FIGURE 25.27 Lysogeny is maintained by an autoregulatory circuit.

The target site on RNA polymerase that the repressor contacts is in the σ⁷⁰ subunit, which is within the region that contacts the –35 region of the promoter. The interaction between the repressor and the polymerase is needed for the polymerase to make the transition from a closed complex to an open complex.

This explains how low levels of repressor positively regulate its own synthesis. As long as enough repressor is available to fill O_R2, RNA polymerase will continue to transcribe the cI gene from P_RM.

25.14 Cooperative Interactions Increase the Sensitivity of Regulation

Lambda repressor dimers interact cooperatively at both the left and right operators, so that their normal condition when occupied by repressor proteins is to have dimers at both the 1 and 2 binding sites. In effect, each operator has a tetramer of repressor. This is not the end of the story, though. The two dimers interact with one another through their C-terminal domains to form an octamer, as depicted in FIGURE 25.28, which shows the distribution of repressors at the operator sites that are occupied in a lysogen. Repressors are occupying O_L1, O_L2, O_R1, and O_R2, and the repressor at the last of these sites is interacting with RNA polymerase, which is initiating transcription at P_RM.

FIGURE 25.28 In the lysogenic state, the repressors bound at O_L1 and O_L2 interact with those bound at O_R1 and O_R2. RNA polymerase is bound at P_RM (which overlaps with O_R3) and interacts with the repressor bound at O_R2.

The interaction between the two operators has several consequences. It stabilizes repressor binding, thereby making it possible for repressor to occupy operators at lower concentrations. Binding at O_R2 stabilizes RNA polymerase binding at P_RM, which enables low concentrations of repressor to autogenously stimulate their own production. The octamer at sites 1 and 2 in O_L and O_R stimulate P_RM transcription better than two dimers at O_R.

The DNA between the O_L and O_R sites (i.e., the gene cI) forms a large loop, which is held together by the repressor octamer. The octamer brings the sites O_L3 and O_R3 into proximity. As a result, two repressor dimers can bind to these sites and interact with one another, as shown in FIGURE 25.29. The occupation of O_R3 prevents RNA polymerase from binding to P_RM, and therefore turns off expression of the repressor.

FIGURE 25.29 O_L3 and O_R3 are brought into proximity by formation of the repressor octamer, and an increase in repressor concentration allows dimers to bind at these sites and to interact.

This shows us how the expression of the cI gene becomes exquisitely sensitive to repressor concentration. At the lowest concentrations, it forms the octamer and activates RNA polymerase in a positive autogenous regulation. An increase in concentration allows binding to O_L3 and O_R3 and turns off transcription in a negative autogenous regulation. The threshold levels of repressor that are required for each of these events are reduced by the cooperative interactions, which make the overall regulatory system much more sensitive. Any change in repressor level triggers the appropriate regulatory response to restore the lysogenic level.

The overall level of repressor has been reduced (about threefold from the level that would be required if there were no cooperative effects), and thus there is less repressor that has to be eliminated when it becomes necessary to induce the phage. This increases the efficiency of induction.

25.15 The cII and cIII Genes Are Needed to Establish Lysogeny

The control circuit for maintaining lysogeny presents a paradox. The presence of repressor protein is necessary for its own synthesis. This explains how the lysogenic condition is perpetuated. How, though, is the synthesis of repressor established in the first place?

When a lambda DNA enters a new host cell, RNA polymerase cannot transcribe cI because there is no repressor present to aid its binding at P_RM. This same absence of repressor, however, means that P_R and P_L are available. Thus, the first event after lambda DNA infects a bacterium is when genes N and cro are transcribed. After this, pN allows transcription to be extended farther. This allows cIII (and other genes) to be transcribed on the left, whereas cII (and other genes) are transcribed on the right (see Figure 25.14).

The cII and cIII genes share with cI the property that mutations in them hinder lytic development. They differ, however, in that the cI mutants can neither establish nor maintain lysogeny. The cII or cIII mutants have some difficulty in establishing lysogeny, but once it is established they are able to maintain it by the cI autoregulatory circuit.

This implicates the cII and cIII genes as positive regulators whose products are needed for an alternative system for repressor synthesis. The system is needed only to initiate the expression of cI in order to circumvent the inability of the autoregulatory circuit to engage in de novo synthesis. They are not needed for continued expression.

The cII protein acts directly on gene expression as a positive regulator. Between the cro and cII genes is the second cI promoter, P_RE. This promoter can be recognized by RNA polymerase only in the presence of cII protein, whose action is illustrated in FIGURE 25.30. The cII protein is extremely unstable in vivo, because it is degraded as the result of the activity of a host protein called HflA (where Hfl stands for high-frequency lysogenization). The role of cIII is to protect cII against this degradation.

FIGURE 25.30 Repressor synthesis is established by the action of cII and RNA polymerase at P_RE to initiate transcription that extends from the antisense strand of cro through the cI gene.

Transcription from P_RE promotes lysogeny in two ways. Its direct effect is that cI mRNA is translated into repressor protein. An indirect effect is that transcription proceeds through the cro gene in the “wrong” direction. Thus, the 5′ part of the RNA corresponds to an antisense transcript of cro; in fact, it hybridizes to authentic cro mRNA, which inhibits its translation. This is important because cro expression is needed to enter the lytic cycle (see the section later in this chapter, The Cro Repressor Is Needed for Lytic Infection).

The cI coding region on the P_RE transcript is very efficiently translated, in contrast with the weak translation of the P_RM transcript. In fact, repressor is synthesized approximately seven to eight times more effectively via expression from P_RE than from P_RM. This reflects the fact that the P_RE transcript has an efficient 5′ UTR containing a strong ribosome-binding site, whereas the P_RM transcript is a very poor mRNA (as noted earlier in this chapter in the section Lambda Repressor Maintains an Autoregulatory Circuit).

25.16 A Poor Promoter Requires cII Protein

The P_RE promoter has a poor fit with the consensus at –10 and lacks a consensus sequence at –35. This deficiency explains its dependence on the positive regulator cII. The promoter cannot be transcribed by RNA polymerase alone in vitro, but can be transcribed when cII is added. The regulator binds to a region extending from about –25 to –45. When RNA polymerase is added, an additional region, which extends from –12 to 13, is protected. As shown in FIGURE 25.31, the two proteins bind to overlapping sites.

FIGURE 25.31 RNA polymerase binds to P_RE only in the presence of cII, which controls the region around –35.

The importance of the –35 and –10 regions for promoter function, in spite of their lack of resemblance with the consensus, is indicated by the existence of cy mutations. These have effects similar to those of cII and cIII mutations in preventing the establishment of lysogeny, but they are cis-acting instead of trans-acting. They fall into two groups, cyL and cyR, which are localized at the consensus operator positions of –10 and –35.

The cyL mutations are located around –10 and probably prevent RNA polymerase from recognizing the promoter.

The cyR mutations are located around –35 and fall into two types, which affect either RNA polymerase or cII binding. Mutations in the center of the region do not affect cII binding; presumably they prevent RNA polymerase binding. On either side of this region, mutations in short tetrameric repeats, TTGC, prevent cII from binding. Each base in the tetramer is 10 bp (one helical turn) separated from its homolog in the other tetramer. This means that when cII recognizes the two tetramers it lies on one face of the double helix.

Positive control of a promoter implies that an accessory protein has increased the efficiency with which RNA polymerase initiates transcription. TABLE 25.1 reports that either or both stages of the interaction between promoter and polymerase can be the target for regulation. Initial binding to form a closed complex or its conversion into an open complex can be enhanced.

TABLE 25.1 Positive regulation can influence RNA polymerase at either stage of transcription initiation.

Promoter	Regulator	Polymerase Binding (equilibrium constant K_B)	Closed–Open Conversion (rate constant, k₂)
P_RM	Repressor	No effect	11χ
P_RE	cII	100χ	100χ

25.17 Lysogeny Requires Several Events

How is lysogeny established during an infection? FIGURE 25.32 recapitulates the early stages and shows what happens as the result of expression of cIII and cII. cIII protects cII from proteolytic degradation by the protease HflA. The presence of cII allows P_RE to be used for transcription extending through cI. Lambda repressor protein is synthesized in high amounts from this transcript and immediately binds to O_L and O_R, initially as monomers, but as the concentration builds up monomers form dimers from P_L/O_L to P_R/O_R, causing a DNA loop to form, as seen in Figures 25.28 and 25.29.

FIGURE 25.32 A cascade is needed to establish lysogeny, but then this circuit is switched off and replaced by the autogenous repressor-maintenance circuit.

By directly inhibiting any further transcription from P_L and P_R, repressor binding turns off the expression of all phage genes. This halts the synthesis of cII and cIII proteins, which are unstable; they decay rapidly, with the result that P_RE can no longer be used. Thus, the synthesis of repressor via the establishment circuit is brought to a halt.

The lambda repressor is now present at O_R2, though. Acting as a positive regulator, it switches on the maintenance circuit for expression from P_RM by making contact with the RNA polymerase sigma factor. This may be a redundant mechanism, simply to ensure the switch. Repressor continues to be synthesized, although at the lower level typical of P_RM function. Thus, the establishment circuit starts off repressor synthesis at a high level, and then the repressor turns off all other functions while at the same time turning on the maintenance circuit, which functions at the low level adequate to sustain lysogeny. At even higher levels of lambda repressor, with occupancy of O_R3, lambda repressor turns off its own synthesis.

Without going into detail on the other functions needed to establish lysogeny, note that the infecting lambda DNA must be inserted into the bacterial genome, aided by its host, which transports the insertion site to lambda near its point of entry (see the chapter titled Homologous and Site-Specific Recombination). The insertion requires the product of the int gene, which is expressed from its own promoter P_I, at which the cII positive regulator also is necessary. The functions necessary for establishing the lysogenic control circuit are therefore under the same control as the function needed to integrate the phage DNA into the bacterial genome. Thus, the establishment of lysogeny is under a control that ensures that all the necessary events occur with the same timing.

Emphasizing the tricky quality of lambda’s intricate cascade, note that cII promotes lysogeny in another, indirect manner. It sponsors transcription from a promoter called P_anti-Q, which is located within the Q gene. This transcript is an antisense version of the Q region, and it hybridizes with Q mRNA to prevent translation of Q protein, whose synthesis is essential for lytic development. Thus, the same mechanisms that directly promote lysogeny by causing transcription of the cI repressor gene also indirectly help lysogeny by inhibiting the expression of cro (described earlier) and Q, the regulator genes needed for the antagonistic lytic pathway.

25.18 The Cro Repressor Is Needed for Lytic Infection

Lambda is a temperate virus; thus it has the alternatives of entering either the lysogenic pathway or the lytic pathway. Lysogeny is initiated by establishing an autoregulatory maintenance circuit that inhibits the entire lytic cascade through applying pressure at two points, P_L O_L and P_R O_R. The two pathways begin exactly the same way—with the immediate early gene expression of the N gene and the cro gene, followed by the pN-directed delayed early transcription. A problem now emerges: How does the phage enter the lytic cycle?

The key to the lytic cycle is the role of the gene cro, which codes for another repressor protein: Cro is responsible for preventing the synthesis of the lambda repressor protein cI. This action shuts off the possibility of establishing lysogeny. Cro mutants usually establish lysogeny rather than entering the lytic pathway, because they lack the ability to switch events away from the expression of repressor.

Cro forms a small dimer (the monomer is 9 kD) that acts within the immunity region. It has two effects:

It prevents the synthesis of the lambda repressor via the maintenance circuit; that is, it prevents transcription via P_RM.
It also inhibits the expression of early genes from both P_L and P_R.

This means that when a phage enters the lytic pathway, Cro has responsibility both for preventing the synthesis of the lambda repressor and subsequently for turning down the expression of the early genes once enough product has been made.

Note that Cro achieves its function by binding to the same operators as the lambda repressor protein, cI. Cro contains a region with the same general structure as the lambda repressor; a helix-2 is offset at an angle from the recognition helix-3. The remainder of the structure is different, which demonstrates that the helix-turn-helix motif can operate within various contexts. As does the lambda repressor, Cro binds symmetrically at the operators.

The sequence of Cro and the lambda repressor in the helix-turn-helix region are related, which explains their ability to contact the same DNA sequence (see Figure 25.22). Cro makes similar contacts to those made by the lambda repressor but binds to only one face of DNA; it lacks the N-terminal arms by which the lambda repressor reaches around to the other side.

How can two proteins have the same sites of action yet have such opposite effects? The answer lies in the different affinities that each protein has for the individual binding sites within the operators. Consider O_R, about which more is known, and where Cro exerts both its effects. The series of events is illustrated in FIGURE 25.33. (Note that the first two stages are identical to those of the lysogenic circuit shown in Figure 25.32.)

FIGURE 25.33 The lytic cascade requires Cro protein, which directly prevents repressor maintenance via P_RM, as well as turning off delayed early gene expression, indirectly preventing repressor establishment.

The affinity of Cro for O_R3 is greater than its affinity for O_R2 or O_R1. Thus, it binds first to O_R3. This inhibits RNA polymerase from binding to P_RM. As a result, Cro’s first action is to prevent the maintenance circuit for lysogeny from coming into play.

Cro then binds to O_R2 or O_R1. Its affinity for these sites is similar, and there is no cooperative effect. Its presence at either site is sufficient to prevent RNA polymerase from using P_R. This, in turn, stops the production of the early functions (including Cro itself). As a result of cII’s instability, any use of P_RE is brought to a halt. Thus, the two actions of Cro together block all production of the lambda repressor.

As far as the lytic cycle is concerned, Cro turns down (although it does not completely eliminate) the expression of the early genes. Its incomplete effect is explained by its affinity for O_R1 and O_R2, which is about eight times lower than that of the lambda repressor. This effect of Cro does not occur until the early genes have become more or less superfluous, because the pQ protein is present; by this time, the phage has started late gene expression and is concentrating on the production of progeny phage particles.

Note that in the early stages of the infection, Cro is given a head start over the lambda repressor, and so it would seem that the lytic pathway is favored. Ultimately, the outcome is determined by the concentration of the two proteins and their intrinsic DNA-binding affinities.

25.19 What Determines the Balance Between Lysogeny and the Lytic Cycle?

The programs for the lysogenic and lytic pathways are so intimately related that it is impossible to predict the fate of an individual phage genome when it enters a new host bacterium. Will the antagonism between the lambda repressor and Cro be resolved by establishing the autoregulatory maintenance circuit shown in Figure 25.32, or by turning off lambda repressor synthesis and entering the late stage of development shown in Figure 25.33?

The same pathway is followed in both cases right up to the brink of decision. Both involve the expression of the immediate early genes and extension into the delayed early genes. The difference between them comes down to the question of whether the lambda repressor or Cro will obtain occupancy of the two operators O_L and P_L.

The early phase during which the decision is made is limited in duration in either case. No matter which pathway the phage follows, expression of all early genes will be prevented as P_L and P_R are repressed and, as a consequence of the disappearance of cII and cIII, production of repressor via P_RE will cease.

The critical question comes down to whether the cessation of transcription from P_RE is followed by activation of P_RM and the establishment of lysogeny, or whether P_RM fails to become active and the pQ regulator commits the phage to lytic development. FIGURE 25.34 shows the critical stage at which both repressor and Cro are being synthesized. This is determined by how much lambda repressor was made. This, in turn, is determined by how much cII transcription factor was made. Finally, this, in turn, is—at least partly—determined by how much cIII protein was made.

FIGURE 25.34 The critical stage in deciding between lysogeny and lysis is when delayed early genes are being expressed. If cII causes sufficient synthesis of repressor, lysogeny will result because repressor occupies the operators. Otherwise Cro occupies the operators, resulting in a lytic cycle.

The initial event in establishing lysogeny is the binding of lambda repressor at O_L1 and O_R1. Binding at the first sites is rapidly succeeded by cooperative binding of further repressor dimers at O_L2 and O_R2. This shuts off the synthesis of Cro and starts up the synthesis of lambda repressor via P_RM.

The initial event in entering the lytic cycle is the binding of Cro at O_R3. This stops the lysogenic maintenance circuit from starting up at P_RM. Cro must then bind to O_R1 or O_R2, and to O_L1 or O_L2, to turn down early gene expression. By halting production of cII and cIII, this action leads to the cessation of lambda repressor synthesis via P_RE. The shutoff of lambda repressor establishment occurs when the unstable cII and cIII proteins decay.

The critical influence over the switch between lysogeny and lysis is how much cII protein is made. If cII is abundant, synthesis of repressor via the establishment promoter is effective, and, as a result, the lambda repressor gains occupancy of the operators. If cII is not abundant, lambda repressor establishment fails, and Cro binds to the operators.

The level of cII protein under any particular set of circumstances determines the outcome of an infection. Mutations that increase the stability of cII increase the frequency of lysogenization. Such mutations occur in cII itself or in other genes. The cause of cII’s instability is its susceptibility to degradation by host proteases. Its level in the cell is influenced by cIII as well as by host functions.

The effect of the lambda protein cIII is secondary: It helps to protect cII against degradation. The presence of cIII does not guarantee the survival of cII; however, in the absence of cIII, cII is virtually always inactivated.

Host gene products act on this pathway. Mutations in the host genes hflA and hflB increase lysogeny. The mutations stabilize cII because they inactivate host protease(s) that degrade it.

The influence of the host cell on the level of cII provides a route for the bacterium to interfere with the decision-making process. For example, host proteases that degrade cII are activated by growth on rich medium. Thus, lambda tends to lyse cells that are growing well but is more likely to enter lysogeny on cells that are starving (and that lack components necessary for efficient lytic growth).

A different picture is seen if multiple phages infect a bacterium. Several parameters are altered. First, more cIII per bacterial cell is made to counter the amount of host protease, and that allows more cII to be made. On the other hand, in a single cell infected by multiple phages each lambda genome will ultimately make its own decision about entering the lytic pathway or lysogenic pathway. This is a “noisy” decision that can be affected by minor local differences in the concentration of different molecules and proteins. The final outcome for the cell is quite different from that of a single-phage infection because the status of each individual phage must be considered. Ultimately, one can imagine that a vote will be taken, and for lysogeny to occur the vote must be unanimous. Even if only one phage proceeds down the lytic pathway, cell death will occur.

Summary

Virulent phages follow a lytic life cycle, in which infection of a host bacterium is followed by production of a large number of phage particles, lysis of the cell, and release of the viruses. Temperate phages can follow the lytic pathway or the lysogenic pathway, in which the phage genome is integrated into the bacterial chromosome and is inherited in this inert, latent form like any other bacterial gene.

In general, lytic infection can be described as falling into three phases. In the first phase a small number of phage genes are transcribed by the host RNA polymerase. One or more of these genes is a regulator that controls expression of the group of genes expressed in the second phase. The pattern is repeated in the second phase, when one or more genes is a regulator needed for expression of the genes of the third phase. Genes active during the first two phases encode enzymes needed to reproduce phage DNA; genes of the final phase code for structural components of the phage particle. It is common for the very early genes to be turned off during the later phases.

In phage lambda, the genes are organized into groups whose expression is controlled by individual regulatory events. The immediate early gene N codes for an antiterminator that allows transcription of the leftward and rightward groups of delayed early genes from the early promoters P_R and P_L. The delayed early gene Q has a similar antitermination function that allows transcription of all late genes from the promoter P_R′. The lytic cycle is repressed, and the lysogenic state maintained, by expression of the cI gene, whose product is a repressor protein, the lambda repressor, that acts at the operators O_R and O_L to prevent use of the promoters P_R and P_L, respectively. A lysogenic phage genome expresses only the cI gene from its promoter, P_RM. Transcription from this promoter involves positive autoregulation, in which repressor bound at O_R activates RNA polymerase at P_RM.

Each operator consists of three binding sites for the lambda repressor. Each site is palindromic, consisting of symmetrical half-sites. Lambda repressor functions as a dimer. Each half-binding site is contacted by a repressor monomer. The N-terminal domain of repressor contains a helix-turn-helix motif that contacts DNA. Helix-3 is the recognition helix and is responsible for making specific contacts with base pairs in the operator. Helix-2 is involved in positioning helix-3; it is also involved in contacting RNA polymerase at P_RM. The C-terminal domain is required for dimerization. Induction is caused by cleavage between the N- and C-terminal domains, which prevents the DNA-binding regions from functioning in dimeric form, thereby reducing their affinity for DNA and making it impossible to maintain lysogeny. Lambda repressor–operator binding is cooperative, so that once one dimer has bound to the first site, a second dimer binds more readily to the adjacent site.

The helix-turn-helix motif is used by other DNA-binding proteins, including lambda Cro. Cro binds to the same operators but has a different affinity for the individual operator sites, which are determined by the sequence of helix-3. Cro binds individually to operator sites, starting with O_R3, in a noncooperative manner. It is needed for progression through the lytic cycle. Its binding to O_R3 first prevents synthesis of repressor from P_RM, and then its binding to O_R2 and O_R1 prevents continued expression of early genes, an effect also seen in its binding to O_L1 and O_L2.

Establishment of lambda repressor synthesis requires use of the promoter P_RE, which is activated by the product of the cII gene. The product of cIII is required to stabilize the cII product against degradation. By turning off cII and cIII expression, Cro acts to prevent lysogeny. By turning off all transcription except that of its own gene, the repressor acts to prevent the lytic cycle. The choice between lysis and lysogeny depends on whether repressor or Cro gains occupancy of the operators in a particular infection. The stability of cII protein in the infected cell is a primary determinant of the outcome.

References

25.4 Two Types of Regulatory Events Control the Lytic Cascade

Review

Greenblatt, J., Nodwell, J. R., and Mason, S. W. (1993). Transcriptional antitermination. Nature 364, 401–406.

25.6 Lambda Immediate Early and Delayed Early Genes Are Needed for Both Lysogeny and the Lytic Cycle

Review

Ptashne, M. (2004). The genetic switch: Phage lambda revisited. Cold Spring Harbor, NY: Cold Spring Harbor Press.

25.8 Lysogeny Is Maintained by the Lambda Repressor Protein

Research

Pirrotta, V., Chadwick, P., and Ptashne, M. (1970). Active form of two coliphage repressors. Nature 227, 41–44.
Ptashne, M. (1967). Isolation of the lambda phage repressor. Proc. Natl. Acad. Sci. USA 57, 306–313.
Ptashne, M. (1967). Specific binding of the lambda phage repressor to lambda DNA. Nature 214, 232–234.

25.9 The Lambda Repressor and Its Operators Define the Immunity Region

Review

Friedman, D. I., and Gottesman, M. (1982). Lambda II. Cambridge, MA: Cell Press.

25.10 The DNA-Binding Form of the Lambda Repressor Is a Dimer

Research

Pabo, C. O., and Lewis, M. (1982). The operator-binding domain of lambda repressor: structure and DNA recognition. Nature 298, 443–447.

25.11 The Lambda Repressor Uses a Helix-Turn-Helix Motif to Bind DNA

Research

Brennan, R. G., Roderick, S. L., Takeda, Y., and Matthews, B. W. (1990). Protein-DNA conformational changes in the crystal structure of a lambda Cro-operator complex. Proc. Natl. Acad. Sci. USA 87, 8165–8169.
Sauer, R. T., Yocum, R. R., Doolittle, R. F., Lewis, M., and Pabo, C. O. (1982). Homology among DNA-binding proteins suggests use of a conserved super-secondary structure. Nature 298, 447–451.
Wharton, R. L., Brown, E. L., and Ptashne, M. (1984). Substituting an α-helix switches the sequence specific DNA interactions of a repressor. Cell 38, 361–369.

25.12 Lambda Repressor Dimers Bind Cooperatively to the Operator

Research

Bell, C. E., Frescura, P., Hochschild, A., and Lewis, M. (2000). Crystal structure of the lambda repressor C-terminal domain provides a model for cooperative operator binding. Cell 101, 801–811.
Johnson, A. D., Meyer, B. J., and Ptashne, M. (1979). Interactions between DNA-bound repressors govern regulation by the phage lambda repressor. Proc. Natl. Acad. Sci. USA 76, 5061–5065.

25.13 The Lambda Repressor Maintains an Autoregulatory Circuit

Research

Hochschild, A., Irwin, N., and Ptashne, M. (1983). Repressor structure and the mechanism of positive control. Cell 32, 319–325.
Li, M., Moyle, H., and Susskind, M. M. (1994). Target of the transcriptional activation function of phage lambda cI protein. Science 263, 75–77.
Michalowski, C. B., and Little, J. W. (2005). Positive autoregulation of CI is a dispensable feature of the phage lambda gene regulatory circuitry. J. Bact. 187, 6430–6442.

25.14 Cooperative Interactions Increase the Sensitivity of Regulation

Review

Ptashne, M. (2004). The genetic switch: Phage lambda revisited. Cold Spring Harbor, NY: Cold Spring Harbor Press.

Research

Anderson, L. M., and Yang, H. (2008). DNA looping can enhance lysogenic CI transcription in phage lambda. Proc. Natl. Acad. Sci. USA 105, 5827–5832.
Bell, C. E., and Lewis, M. (2001). Crystal structure of the lambda repressor C-terminal domain octamer. J. Mol. Biol. 314, 1127–1136.
Cui, L., Murchland, I., Shearlin, K. E., and Dodd, I. A. (2013). Enhancer-like long range transcriptional activation by lambda CI- mediated DNA looping. Proc. Natl. Acad. Sci. USA 110, 2922–2928.
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25.17 Lysogeny Requires Several Events

Research

Tal, A., Arbel-Goran, R., Castanino, N., Court, D. L., and Stavans, J. (2014). Location of the unique integration site on an E. coli chromosome by bacteriophage lambda DNA in vivo. Proc. Natl. Acad. Sci. USA 111, 349–354.

25.19 What Determines the Balance Between Lysogeny and the Lytic Cycle?

Review

Oppenheim, A. B., Kobiler, O., Stavans, J., Court, D. L., and Adhya, S. (2005). Switches in bacteriophage lambda development. Annu. Rev. Gen. 39, 409–429.

Research

Zeng, L., Skinner, S. O., Zong, C., Skippy, J., Feiss, M., and Golding, I. (2010). Decision making at a subcellular level determines the outcome of a bacteriophage infection. Cell 141, 682–691.