1. Determination of Phage Cycles Upon Infection of E. coli Mike Gleason University of Nebraska Medical Center

2. 1 Initial Infection intxiscIIINcI CrocIIOP Upon infection, here is what the injected phage genome looks like. All genes are turned off until host transcription machinery binds to the phage DNA.

3. 2 Immediate Early Genes intxiscIIIcI cIIOP PRPR PLPL N Cro The host’s RNA polymerase has now started transcribing from both the P L and P R promoters. This results in production of the antiterminator (N) protein and some Cro protein. At this time, both transcripts terminate early due to the presence of a rho- independent terminator sequence. STOP Products: N, Cro

4. 3 Early Genes intxiscIIIN CrocIIOP N N PRPR PLPL cI Products: N, cIII, xis, int, Cro, cII, O, P The antiterminator (N) protein allows both P L and P R to be read through, producing longer transcripts. Since prokaryotes have polycistronic mRNAs, each longer transcript can now code for multiple proteins.

5. 4 Decision Time intxiscIIIN CrocIIOP N N PRPR PLPL cI Products: N, cIII, xis, int, Cro, cII, O, P The virus now uses the host’s relative health to decide whether to enter the lytic phase or lysogeny. If there is ample glucose available to the host, cAMP is exported and [cAMP] is low. Supplies of cAMP determines whether it can bind and inactivate the host proteolytic enzyme HflA.

6. 5 Lysis CrocIIOP N PRPR PLPL Products: Cro, cII, O, P Glucose was in ample supply, so cAMP is exported and [cAMP] is low. cAMP does not bind HflA, so HflA is free to degrade cII, and therefore not lead to cI. The site between the P R and P RM is the operator OR. It has three similar cis elements, OR1, OR2, and OR3, each of which can be bound by Cro or cI. There are slight differences in the sequences, so Cro has highest affinity for OR3, followed by OR2 and OR1. cI has the opposite affinity; it prefers OR1, then OR2 and OR3. When cI binds at OR1, it prevents RNA Pol from binding at P R. Similarly, Cro binding at OR3 blocks P RM. Therefore, Cro and cI can compete for these binding sites. At lower [cI], Cro is able to outcompete cI at O R and O L and stimulate lysis. At medium levels of [Cro], P L and P RM are disabled, but P R remains active, producing O and P for lysis. Cro P RM intxiscIIINcI N OR3OR2OR1 Cro P RM PRPR

7. 6 Lysis 2 PLPL Products: (None) When [Cro] becomes high, all promoters are shut off. P RM intxiscIIINcI OR3OR2OR1 P RM PRPR PRPR STOP cIIOPCro

8. 7 Decision Time intxiscIIIN CrocIIOP N N PRPR PLPL cI Products: N, cIII, xis, int, Cro, cII, O, P Now let’s see what happens when lysogeny is chosen.

9. 8 Lysogeny intxiscIIIN CrocIIOP N N PRPR PLPL Products: N, cIII, xis, int, Cro, cII, O, P, cI. Glucose was scarce, so cAMP was not exported and [cAMP] is high. cAMP binds HflA, so HflA does not degrade cII. The cIII protein helps protect cII, and together they activate P I and P RE. This results in more int protein, and the cI protein (The Repressor). The P RE promoter that leads to production of cI is a very weak promoter. cII is needed so that RNA Polymerase is able to bind this promoter effectively. The transcript has a Shine-Dalgarno sequence, so it is efficiently translated once it is produced. This helps accelerate initial production of cI. The P RM promoter, which also produces cI, is not yet active. When it is active, it is not efficiently translated (about 8X less than P RE ) because it lacks the Shine-Dalgarno sequence. This promoter is only needed for occasional production of cI (in maintenance mode), so this reduced level of expression is desirable. P RE PIPI cII cIII cII cIII cI P RM

10. 9 Lysogeny 2 PLPL Products: cI At this point, enough proteins for integration into the host genome have been produced, so lysogeny begins. When cI is produced, it shuts off all promoters except P M and maintains lysogeny. It upregulates P M, until [cI] becomes very high, where it then shuts off P M also. intxiscIIIN OR3OR2OR1 P RM PRPR PRPR STOP cIIOPCro cI P RM cI PIPI

11. 10 Induction PLPL Products: cI When the host cell is in danger, its “S.O.S.” response activates the host’s multi-purpose RecA protein. RecA interacts with cI, and cI is cleaved. intxiscIIIN OR3OR2OR1 P RM PRPR PRPR STOP cIIOPCro cI P RM cI RecA

12. 11 Induction 2 CrocIIOP N PRPR PLPL Products: Cro, cII, O, P With RecA degrading any cI that is produced, Cro is able to outcompete cI and begin lysis. Cro P RM intxiscIIINcI N OR3OR2OR1 Cro P RM PRPR

13. 12 References Tapprich, W. University of Nebraska Omaha. (2004) Virology notes. Cox, G. S. University of Nebraska Medical Center. (2006) BRTP 822 handouts. Echols, H., Murialdo, H. (1978) Microbiology Reviews 42:577-591.