Transcription Regulation in Phage

Repressors/Operators - Critical events in transcriptional regulation involve two different repressor proteins, called cI and Cro, each of which binds at two different operators. Each operator contains three repressor binding sites. The repressors bind to each of the six operator sites with varying affinities, leading to varying occupancy of each binding site by each repressor under varying physiological conditions. The cI repressor also serves, under certain conditions, as a transcriptional activator, promoting the expression of some genes while repressing that of others. Cro acts both as a repressor and as an antirepressor, because it antagonizes the action of cI in a very specific way (Figure 26.27). Transcription from the two promoter-operator sites takes place in opposite directions along the genome (Figure 26.23).

The cI Repressor and its Operators - The cI repressor is a dimeric protein, with a subunit Mr of 27,000. It binds through its N-terminal sequences to operator sites with a Ka of about 3 x 10¹³ M (Figure 26.24). Two operators, on either side of the cI gene, control divergent transcriptional events from a central regulatory region-leftward (OL) and rightward (OR). Each operator contains three separate repressor-binding sites, each about 17 base pairs long. The three repressor-binding sites are homologous, but not completely so (Figure 26.26), and they are separated by spacer regions of three to seven base pairs. A fully virulent mutant (one which does not establish a lysogenic state) has at least two mutations-one in OL and one in OR.

Operator features - The operators have the following interesting features:

1. Operators and promoters are interspersed, so the regulatory regions are more properly called OLPL and ORPR;

2. ORPR controls transcription from two distinct promoters-one rightward (PR) and one leftward (PRM) (Figure 26.26);

3. Transcription from OLPL and ORPR is controlled by two different repressors-cI and Cro;

4. Under certain conditions the cI repressor is a transcriptional activator, not an inhibitor; and

5. cI transcription is initiated from different promoters under different physiological conditions.

All of these complexities are related to the need for orderly and efficient phage gene control under quite different physiological conditions.

Interactions Between the Two Repressors - The decision between lytic and lysogenic infection is made at ORPR. It involves interactions of cI and Cro. Of the three repressor-binding sites in ORPR, cI binds most tightly to site OR1, less tightly to OR2, and still less tightly to OR3. cI binding is cooperative, so that when one repressor dimer is bound at OR1, affinity for a second molecule is increased at OR2. Cro protein binds considerably less tightly to any of the sites than does cI and in the reverse order. That is, site OR3 is favored, followed by approximately equal binding at OR2 and OR1. Binding of Cro is noncooperative.

Lysogeny establishment - As lysogeny is being established (Figure 26.27b), when lytic and lysogenic genes are competing to determine the fate of the viral genome, there is a need for larger amounts of cI repressor than can be transcribed from PRM (the 'M' stands for "maintenance," because this is the promoter from which cI is transcribed during maintenance of lysogeny). At this time a cI promoter, called PRE (the 'E' stands for "establishment"), is activated. In this activation the cII protein binds specifically at the -35 region of PRE and stimulates RNA polymerase binding at that site. This transcriptional event yields a longer cI messenger RNA that is more efficiently translated than the message synthesized from PRM. The result is sufficient cI repressor to bind all three sites in OR and, hence, to block both transcriptional initiation events. Because of the cooperative binding of cI to its operators, both sites OR1 and OR2 are usually occupied in the lysogenic state (Figure 26.27a), even though the intracellular concentration of cI is quite low (about 200 molecules per cell, or 10^-7 M). Binding of cI in this manner inhibits the transcription of Cro from its own promoter and activates the leftward transcription of itself from the promoter PRM. The -10 and -35 regions for the PRM promoter lie within the operators.

Lytic infection - In prophage induction, lysogeny is broken, leading to a lytic infection (Figure 26.27c). First, cI repressor is inactivated, and the OR sites become unoccupied. This permits transcription of cro from PR, and the Cro protein blocks further transcription of cI from PRM. At the same time, leftward transcription from PL generates the N protein, blocking transcriptional termination at the sites indicated in Figure 26.27 as tR1 and tL. Thus, the two early transcripts for Cro and N are extended to activate new genes. Leftward transcription generates Int and Xis proteins, necessary for prophage excision. Rightward transcription generates O and P, both of which are necessary for DNA replication.

Subsequent regulatory events, including the action of the gene Q protein, activate transcription of late-acting genes, which encode structural proteins of the virus. At this time it is desirable to suppress early gene transcription, so that the late proteins can be made at maximal rates. This involves further action of the Cro protein, which by this time has accumulated to the point that it can bind to both OR1 and OL1, blocking transcription from PR and PL, respectively (Figure 26.27d). In infection by a virulent mutant (Figure 26.27e), cI transcription from PRM is blocked, and this leads to activation of cro transcription from PR.

Phage Biology

1. Bacteriophage

2. Cro Repressor Image