A genetic switch with memory: the lysis/lysogeny switch in phage  AMATH 882 Lecture 11, Feb. 12, 2013 Reference: A Genetic Switch, by Mark Ptashne

As an example of a genetic switch which can be triggered by a transient stimulus, we consider the case of phage . Phage  is a bacteriophage -- a virus which infects E. coli.

Phage Lambda: structure and infection http://fig.cox.miami.edu/Faculty/Dana/phage.jpg http://de.wikipedia.org/wiki/Bild:T4-phage.jpg

Upon infection, the phage has two mechanisms of action: Lysogeny, in which the phage chromosome is integrated into the host’s genome. The phage then dormantly infects all progeny, as its genome (called the prophage) is replicated when the host divides. Lytic Growth, in which the host’s genetic machinery is used to produce ~100 new phages, and then the host cell is lysed (broken).

The phage “chooses” between these two mechanisms based on a “reading” of the host’s behaviour. If the host is growing well the phage lysogenizes the host and subsequently infects all of its progeny. If the host is not growing well (e.g. starving), the phage grows lytically - an 'abandon ship' response. This “decision” is based on a genetic switch.

The switch is composed of two genes and their products: Gene cI which codes for repressor Gene cro which codes for Cro (control of repressor and others)

These genes are adjacent to one another on the phage genome: PR (the right promoter) is the promoter for cro PRM (repressor maintenance) is the promotor for cI

Both repressor and Cro regulate gene expression by binding to the the right operator OR, which is divided into three operator regions: OR1, OR2 and OR3

Repressor protein folds into two domains (labeled amino (N) and carboxyl (C) for the two ends of the polypeptide chain). The carboxyl domains associate with one another, so that the protein is predominantly present as a dimer.

These repressor dimers bind strongly to OR1 and weakly to OR2 and OR3 (about ten times less affinity).

However, repressor bound to OR1 binds cooperatively to repressor at OR2 greatly increasing affinity for that site. The result: at low to mid concentrations, repressor is bound to OR1 and OR2. Only at much higher levels is it also found bound to OR3.

repressor at OR1 inhibits Cro by blocking the binding site PR Effects of repressor binding: repressor at OR1 inhibits Cro by blocking the binding site PR

Effects of repressor binding: repressor at OR2 upregulates cI by binding to RNA polymerase at PRM, increasing its affinity for the promoter Thus at low-mid levels, repressor increases its own rate of production (positive feedback)

Effects of repressor binding: repressor at OR3 inhibits cI by blocking PRM. Thus at high levels, repressor decreases its own rate of production (negative feedback)

Conclusion: Repressor blocks production of Cro and acts to maintain its own concentration at a particular level. The result is a stable steady state -- repressor high, Cro low.

Cro is a smaller protein than repressor, and folds into a single domain. Like repressor, it is found mainly in the form of a dimer.

Also like repressor, dimers of Cro bind to each of the subregions of the right operator.

However, Cro's affinity for these operator regions is opposite that of repressor. There are no cooperative effects. Affinity for OR1 and OR2 is roughly equal, and is less than that for OR3.

Cro at OR3 inhibits cI by blocking the binding site PRM Effects of Cro binding: Cro at OR3 inhibits cI by blocking the binding site PRM

At higher concentrations, Cro inhibits its own production by blocking RNA polyermase from PR. No upregulation is needed since PR has a much higher affinity for RNA polymerase than does PRM.

Conclusion: Cro blocks production of repressor and acts to maintain its own concentration at a particular level. The result is a stable steady state -- repressor low, Cro high.

Summary: System is bistable, with two stable steady states: lysogeny: repressor levels are high, cro inactive, levels of Cro low. lysis: levels of Cro are high, cI inactive, levels of repressor low. Cro then triggers production of other phage genes needed to continue lytic growth. Question: How does the phage switch from one state to the other?

Hunger (or DNA damage, which can be induced in the lab by exposure to ultraviolet light) causes an increase in the activity of the bacterial (i.e. host) protease RecA. (A protease is an enzyme which degrades proteins). RecA cleaves repressor rendering it unable to dimerize and hence inactive.

Once cleaved, repressor is unable to dimerize and so is inactive -- it cannot bind to DNA to promote its own production or to inhibit production of Cro.

The all-or-nothing, as opposed to graded, behaviour of this induction by UV (or hunger) is a direct consequence of the cooperativity in the repressor mechanism. A hypothetical analog with no cooperativity does not lead to the same switch-like behaviour.

Motivation: such genetic switches play a key role in development (growth of a multicellular organism from a single egg). An an egg divides, each cell receives the same genetic content (i.e. identical copies of DNA). What distinguishes different cell types is the complement of genes which are expressed. Cell differentiation occurs when a cell "chooses" to express a certain gene profile. This "choice" is typically based on signals from the environment. These signals are often the result of gradients of chemical messengers (morphogens), which have been set up as part of development. A switch-like response allows a gradient to set up differentiation into a discrete number of cell types.

morphogen gradient response of genetic network differentiation into two distinct cell types