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Determination of l Phage Cycles Upon Infection of E. coli

Determination of l Phage Cycles Upon Infection of E. coli. Mike Gleason University of Nebraska Medical Center. Initial Infection. Cro. cII. O. P. int. xis. cIII. N. cI.

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Determination of l Phage Cycles Upon Infection of E. coli

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

  2. Initial Infection Cro cII O P int xis cIII N cI Upon infection, here is what the injected l phage genome looks like. All genes are turned off until host transcription machinery binds to the phage DNA.

  3. Immediate Early Genes PR STOP Cro cII O P int xis cIII N cI STOP PL Products: N, Cro The host’s RNA polymerase has now started transcribing from both the PL and PR 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.

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

  5. Decision Time PR N Cro cII O P int xis cIII N cI N PL 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. Lysis PR N Cro cII O P int xis cIII N cI N Cro Cro PR PRM OR3 OR2 OR1 PRM PL Cro Cro 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 PR and PRM 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 PR. Similarly, Cro binding at OR3 blocks PRM. Therefore, Cro and cI can compete for these binding sites. At lower [cI], Cro is able to outcompete cI at OR and OL and stimulate lysis. At medium levels of [Cro], PL and PRM are disabled, but PR remains active, producing O and P for lysis.

  7. Lysis 2 PR STOP Cro cII O P int xis cIII N cI STOP Cro Cro PR PRM OR3 OR2 OR1 PRM PL Cro Cro Cro Products: (None) When [Cro] becomes high, all promoters are shut off.

  8. Decision Time PR N Cro cII O P int xis cIII N cI N PL Products: N, cIII, xis, int, Cro, cII, O, P Now let’s see what happens when lysogeny is chosen.

  9. Lysogeny PR N Cro cII O P int xis cIII N cI N PI PRE PRM cII cII PL cIII cIII 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 PI and PRE. This results in more int protein, and the cI protein (The l Repressor). The PRE 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 PRM promoter, which also produces cI, is not yet active. When it is active, it is not efficiently translated (about 8X less than PRE) 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.

  10. Lysogeny 2 cI PR STOP Cro cII O P int xis cIII N cI PRM STOP cI PI PR OR3 OR2 OR1 PRM PL cI cI 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 PM and maintains lysogeny. It upregulates PM, until [cI] becomes very high, where it then shuts off PM also.

  11. Induction RecA cI PR STOP Cro cII O P int xis cIII N cI PRM STOP cI PR OR3 OR2 OR1 PRM PL RecA cI cI Products: cI RecA RecA 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.

  12. Induction 2 PR N Cro cII O P int xis cIII N cI N Cro Cro PR PRM OR3 OR2 OR1 PRM PL Cro Cro Products: Cro, cII, O, P With RecA degrading any cI that is produced, Cro is able to outcompete cI and begin lysis.

  13. 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 Reviews42:577-591.

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