Genes VII

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. But how 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. But this same absence of repressor means that P_R and P_L are available. So the first event when lambda DNA infects a bacterium is for genes N and cro to be transcribed. Then pN allows transcription to be extended farther. This allows cIII (and other genes) to be transcribed on the left, while cII (and other genes) are transcribed on the right (see Figure 11.12).

The cII and cIII genes share with cI the property that mutations in them cause clear plaques. But there is a difference. The cI mutants can neither establish nor maintain lysogeny. The cII or cIII mutants have some difficulty in establishing lysogeny, but once established, they are able to maintain it by the cI autogenous 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 autogenous circuit to engage in de novo synthesis.

The CII protein acts directly on gene expression. Between the cro and cII genes is another promoter, called P_RE. (The subscript "RE" stands for repressor establishment.) This promoter can be recognized by RNA polymerase only in the presence of CII, whose action is illustrated in Figure 11.23.

The CII protein is extremely unstable in vivo, because it is degraded as the result of the activity of a host protein called HflA. The role of CIII is to protect CII against this degradation.

Transcription from P_RE promotes lysogeny in two ways. Its direct effect is that cI is translated into repressor protein. An indirect effect is that transcription proceeds through the cro gene in the "wrong" direction. So the 5′ part of the RNA corresponds to an antisense transcript of cro; in fact, it hybridizes to authentic cro mRNA, inhibiting its translation. We see in the next section that this is important because cro expression is needed to enter the lytic cycle.

The cI coding region on the P_RE transcript is very efficiently translated (in contrast with the weak translation of the P_RM transcript mentioned earlier). In fact, repressor is synthesized ~7 V8 times more effectively via expression from P_RE than from P_RM. This reflects the fact that the P_RE transcript has an efficient ribosome-binding site, whereas the P_RM transcript has no ribosome-binding site and actually starts with the AUG initiation codon.

The P_RE promoter has a poor fit with the consensus at V10 and lacks a consensus sequence at V35. This deficiency explains its dependence on 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 V25 to V45. When RNA polymerase is added, an additional region is protected, extending from V12 to +13. As summarized in Figure 11.24, the two proteins bind to overlapping sites.

The importance of the V35 and V10 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.

The cyL mutations are located around V10, and probably prevent RNA polymerase from recognizing the promoter.

The cyR mutations are located around V35, and fall into two types, affecting 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, so 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. Figure 11.25 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.

Now we can see how lysogeny is established during an infection. Figure 11.26 recapitulates the early stages and shows what happens as the result of expression of cIII and cII. The presence of CII allows P_RE to be used for transcription extending through cI. Repressor protein is synthesized in high amounts from this transcript. Immediately it binds to O_L and O_R.

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, which are unstable; they decay rapidly, with the result that P_RE can no longer be used. So the synthesis of repressor via the establishment circuit is brought to a halt.

But repressor now is present at O_R. It switches on the maintenance circuit for expression from P_RM. Repressor continues to be synthesized, although at the lower level typical of P_RM function. So the establishment circuit starts off repressor synthesis at a high level; then 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.

We shall not now deal in detail with the other functions needed to establish lysogeny, but we can just briefly remark that the infecting lambda DNA must be inserted into the bacterial genome (see 14 Recombination and repair). The insertion requires the product of gene int, which is expressed from its own promoter P_I, at which CII also is necessary. The sequence of P_I shows homology with P_RE in the CII binding site (although not in the V10 region). The functions necessary for establishing the lysogenic control circuit are therefore under the same control as the function needed physically to manipulate the DNA. So the establishment of lysogeny is under a control that ensures all the necessary events occur with the same timing.

Emphasizing the tricky quality of lambda’s intricate cascade, we now know 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. So 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 (see above) and Q, the regulator genes needed for the antagonistic lytic pathway.