Lytic cascade in lambda (λ) phage

Lambda (λ) bacteriophage exhibits one of the most interesting, but intricate cascade circuits for the two alternative pathways (lytic cycle or lysogeny), that it can follow after infection. A brief mention of this phage and its mode of infection was earlier made in Sexuality and Recombination in Bacteria and Viruses, where it was also explained that the temperate phages like lambda can be classified as episomes, since these are dispensible and exist in free as well as integrated states. The phage can infect bacteria and multiply in the normal manner leading to lysis, but this mode can shift to lysogeny where the phage DNA gets integrated with host DNA and stays tnere 'as prophage till again it becomes lytic. "How are these two states interconnected, and how does one state shift to another" are the subject of regulation of gene expression. A map of lambda (λ)DNA is given in Figjire 36.2, which has early genes in the middle and late genes at both ends of linear DNA. The linear DNA takes a circular shape during infection, so that the late genes from both ends come together in a cluster to form one transcription unit. When the phage infects a host cell, the early and delayed early genes of the phage are expressed irrespective of whether the phage has to follow lytic or lysogenic mode of development. At this stage, a decision about alternative pathways of development is taken. If the late genes are expressed at this stage, lytic cycle sets in, but if^a regulator gene (cI) synthesizing a repressor is expressed, the late genes can not be expressed and lysogeny sets in (Fig. 36.3). It is interesting to note that repressor protein coded by cI regulates its own synthesis also by working as an activator, so that if repressor is inactivated, its further synthesis does not take place and the phage is forced to enter a lytic cycle. Mutants in the gene cI cannot maintain lysogeny, since then the late' genes can not be stopped from being expressed, due to the absence of active repressor.

Three sets of genes in lambda (λ)phage Immediate early cro and N genes and their control region (P_R/O_R and P_L/O_L). As soon as the phage infects, transcription is initiated with the help of two promoters, P_R on the right side and P_L on the left side. The transcription starts on both DNA strands, one strand being transcribed on the left side and the other on the right side. There are two immediate early genes cro and N, cro being transcribed on the right side under the control of P_R and N being transcribed on the left side under the control of P_L. When transcribed, cro has dual function, namely (i) it prevents synthesis of repressor by cI (an action which is necessary for lytic cycle), which otherwise causes lysogeny and (ii) it turns off the expression of the immediate early genes including its ownself, since their expression is not needed later in the lytic cycle. Gene N, as earlier discussed in Expression of Gene : Protein Synthesis 2.  Transcription in Prokaryotes and Eukaryotes, codes for an antiterminator pN (pN = product of gene N), which allows transcription to proceed into the delayed early genes without the need of any fresh promoter or operator genes. Associated with promoters P_R and P_L are operators O_R and O_L respectively which are sites for repressor binding to prevent RNA polymerase from initiating transcription (Fig. 36.2). The gene cI (regulator gene coding for repressor) lies in between-P_R/O_R and P_L/O_L control regions and when active, it not only inhibits the expression of cro and N but also promotes its own expression (Fig. 36.4), so that the repressor has a dual function and also works as an activator for its own autogenous expression and regulation.

Delayed early genes (cIIcIII and Q)and their control on lysogeny and lytic cycle. Among the delayed early genes, regulators cII (on the right) and cIII (on the left) regulate synthesis of repressor by cl, to allow phage enter lysogeny (Figs. 36.2, 36.3). On the other hand, a gene Q (on the right) is a regulator which acts as an antiterminator, and allows the transcription of late genes meant for lysis. The two pathways (lytic and lysogenic) are so intimately related that it is difficult to predict which pathway will be followed and how is the decision for two alternative pathways is actually taken. This will be further discussed later in this section.

Late genes for lysis (right end) and, tail and head genes (left end). In the phage particle, the late genes are found at the two ends of the linear DNA molecule, genes 5 and R on the right end, and the tail and head genes on the left end. Genes for recombination are also found on the left side of cIII and are transcribed as late genes. Soon after the infection, the two ends of DNA molecule join to form a ring, so that all late genes are arranged in a single group containing S-R genes from right end and head and tail genes A to J from left end (Fig. 36.2).

There is a promoter gene P_R’ between genes Q and S. In the absence of pQ (product of Q gene), the transcription from P_R’ is constitutive, but terminates at t_R3 lying close to P_R’ giving a product, 6S RNA (194 bases long). However, when pQ is present, it suppresses t_R3 and 6S RNA is extended with the result that late genes are heavily expressed. Thus, Q gene induces transcription of late genes, which continue to be transcribed all through their length. On the left side also, late genes continue to be transcribed. Transcription from both sides stops on the ring molecule, before the RNA polymerase molecules from two sides could clash.

Anti-repressor (product of cro gene) for lytic cycle
We have earlier discussed that cro gene has dual function of preventing the synthesis of repressor and of turning off the early genes. It codes for a protein (9000 daltons) which forms a dimer and acts on promoter P_M (for cI gene), P_L (for N gene on the left side) and P_R (for cro gene) itself, so that it stops expression of repressor gene cI as well as that of the early genes (Figs. 36.3, 36.5) including N on the left and cro on the right. This fulfils the early requirement for establishment of lytic cycle, because if cI is expressed, this will stop expression of late genes and thus will establish lysogeny, which is discussed in the next section.

Lysogeny through establishment and maintenance of repressor (promoters P_E and P_M)
In phage lambda (λ), a very delicate balance is maintained between lytic cycle and lysogeny, with a very sophisticated and intricate circuit of events, which will be briefly described in this section. It will be seen that the repressor's synthesis is first established with the help of a promoter P_E under the control of genes cII and cIII, and is subsequently maintained by the repressor itself through a promoter P_M.

Promoter P_E (or P_RE)for repressor establishment and the clear plaque genes (cI, cII, cIII). There is a promoter gene P_E lying between cro and cII genes. The product of genes cII and cIII act on the promoter P_E, so that the RNA polymerase ' enzyme can initiate transcription towards the left thus synthesizing RNA on genes cro and cI. This transcript will consist of (i) the transcript from the antisense strand of cro gene, which can not be translated (cro gene is transcribed in the right direction from P_R to yield translatable mRNA) and (ii) mRNA from cl gene which can be translated to give rise to repressor (Fig. 36.6). The efficiency of synthesis of repressor from cI through P_E is 7-8 times the synthesis from the promoter P_M (see next section for further details). This repressor synthesized under the control of P_E, binds immediately to the operators O_L and O_R thus inhibiting transcription from P_L and P_R, leading to turning off the expression of all phage genes. This also halts the synthesis of cII and cIII proteins. Since cII and cIII proteins are unstable and rapidly decay, P_E can not be used any longer for synthesis of repressor. The repressor is now synthesized under the influence of P_M due to the positive control of repressor (already synthesized under the control of promoter P_E), which binds on O_R (which also contains the promoter P_R).

The genes cI, cII and cIII are so named, because mutants in these genes produce clear plaques, due to the presence of only lysed cells. In the wild type phage, when the lysis occurs, due to the presence of some lysogenic bacteria, clear plaques are not obtained (plaque is a region on the culture medium on the plate, generated due to lysis of bacterial cells). Mutants in cI (cI^-)differ from the mutants cII^- and cIII^-, since the former can neither establish, nor maintain lysogeny. The mutants cII^- and cIII^- have difficulty in establishing lysogeny, but once established, lysogeny can be maintained. This suggested that the products of genes cII and cIII are needed only for the establishment of lysogeny, but not for its maintenance. The genes cII and cIII are thus needed to circumvent the difficulty of autogenous circuit of cI, which can not start the synthesis of repressor de novo. The different steps involved in establishment and autogenous maintenance of lysogeny are shown in Figure 36.7.

Promoter P_M (P_RM)for autogenous maintenance of repressor. Once the synthesis of repressor is established through the action of cII and cIII gene products on P_E , these genes (cII and cIII)are switched off by the action of repressor on O_R and O_L. But the repressor works as an activator by binding on O_R, which also contains the promoter P_M (also known P_RM since it is on the right side of cI). By acting on O_R, the repressor helps RNA polymerase to bind on P_M and start further synthesis of repressor. In a remarkable manner it also controls the concentration of repressor, so that when repressor concentration is too high, its further synthesis can be stopped. This is discussed in the following section.

Autoregulation of repressor synthesis by cooperative binding of repressor. Each operator (O_R and O_L)has three binding sites, O_R with O_R1-O_R2-O_R3and O_L with O_Ll-O_L2-O_L3. In each case, site 1 is closest to its promoter (P_R and P_L)and site 3 is farthest. Site 1 has greater affinity (roughly tenfold) for repressor than other sites in each operator, so that the repressor first binds to O_R1and O_L1. The repressor functions as dimers for maintenance of lysogeny, and at low concentration it binds only to sites O_R1and O_L1, while with increasing concentration, the repressor dimers can also bind to sites 2(O_R2 and O_L2)and 3(O_R3 and O_L3). Presence of repressor on site 1 increases its affinity for site 2 but this interaction does not proceed to site 3. When lysogeny is established, only sites 1 and 2 are occupied but site 3 is not occupied. Since P_R lies within O_R1and P_L lies within O_L1, occupancy of O_R1-O_R2 and O_L1-O_L2 physically block P_R and P_L respectively and inhibit the expression of all genes thus establishing lysogeny.

The relationship of O_R and P_M is such that P_M (site for RNA polymerase binding) lies rather close to O_R2. When dimers are bound to O_L1-O,2, the dimer at O_R2 interacts with RNA polymerase in such a way that the latter can utilize P_M for transcription (Fig. 36.8). It is interesting that a repressor dimer bound on O_R2 can inhibit transcription from P_R, but promote transcription from P_M.

However, when repressor concentration increases to such an extent, that it may now occupy even I_R3 in addition to O_R1-O_R2, the transcription from cI is prevented. This effect depends on the fact that P_M lies within O_R3 and when O_R3 is occupied by repressor, it is not available for RNA polymerase. When repressor synthesis stops due to occupancy of O_R3 by repressor, the concentration of repressor consequently falls, and O_R3 again becomes available for RNA polymerase and synthesis of repressor starts once again due to the presence of repressor on O_R2. Thus repressor is an autogenous regulator of its own synthesis causing the inhibition by negative control at its high concentration and promoting by positive control at its low concentration.

Change from lysogeny to lytic cycle by cleavage of repressor dimers in the connector region. The repressor functions as a dimer and each unit (27,000 daltons) has (i) a N-terminal domain (1-92 residues), which binds to operator; (ii) a C-terminal end (133-132 residues), which enhances the affinity of N-terminal domain for binding and (iii) a connector region (93-132 residues). The induction of a lysogenic prophage to enter the lytic cycle is caused by cleavage of repressor in the connector region between 111 and 112 amino acid residues. The cleavage releases the C-terminal domains, so that N-terminal domains now do not have sufficient affinity to remain attached to the operator. N-terminal domains thus dissociate, leading to lytic infection, because now the genes repressed earlier due to repressor, will be expressed causing lysis (Fig. 36.9). This transition from lysogeny to lysis, involves the expression of Q gene with the help of promoter P_R' giving its product pQ regulator which helps in the expression of late genes S and R (Fig. 36.5).