Monday, November 4th Lecture Notes

  • If cI can establish autoregulation, cI “permanently” represses cro

  • But if cI fails to establish autoregulation, cro successfully represses cI

  • How does the phage choose which pathway to follow

    • cII acts as a sensor for environmental factors

  • cII

    • The function of the cII gene is to initiate the transcription of cI from the PRE promoter

    • Without help from cII, the cI gene will not be able to compete with cro and the phage will always choose the lytic pathway

  • The decision between lysogeny and lysis is heavily influenced by the state of the bacterial host cell:

    • When E. coli is healthy and actively dividing, it has large quantities of a protease (FtsH) that degrades the cII protein

      • This gives Cro the upper hand and favors lysis

    • But if the infected bacterium is growing slowly, it contains little FtsH and cII protein is relatively stable

      • This gives cI the upper hand and favors lysogeny

    • Because the FtsH protease degrades the phage protein cII, it can prevent cI expression

    • As a result, Cro protein dominates and the phage enters the lytic pathway

    • With little or no FtsH, the cII activates transcription from PRE and synthesis of cI protein

      • This gives cI a chance to establish positive autoregulation, which can repress cro and favor lysogeny

  • Pros and Cons

    • Lytic cycle

      • Maximizes the rate of reproduction

      • Most favorable in a dense, rapidly growing population of bacteria because there are new hosts available

    • Lysogeny

      • Results in slower reproduction but it optimizes survival

      • Prophage is replicated when the host bacterium divides

      • Protected by the host cell

      • Safer strategy in sparse populations of slowly growing bacteria

  • What happens if more than one phage infects a given cell?

    • Each phage synthesizes cII protein independently

    • The bacterium contains a fixed amount of FtsH, so an extra dose of cII will favor lysogeny

    • However, typically only 1 phage genome can actually integrate into the bacterial chromosome

  • If the phage commits to lysogeny, it will express lambda integrase that inserts the phage DNA into the bacterial chromosome

  • Recombinases

    • Lambda integrase is an example

    • Functions:

      • Bring together two DNA molecules or two different sites on the same molecule

      • Cleave phosphodiester bonds in both strands of each DNA sequence

      • Religate those cleaved ends into a new configuration

  • Classes of Recombination

    • Homologous recombination

      • Can occur between any two DNA sequences that share more than 50 bp of nearly identical sequence

      • Examples: repair of double-stranded breaks, meiotic crossover

    • Conservative site-specific recombination

      • Occurs at 2 specific, pre-defined DNA sequences selected by evolution

      • Examples: lysogeny of lambda phage, rearrangement of immunoglobulin genes

    • Transposition

      • Occurs when one specific, pre-defined DNA sequence inserts itself into another DNA molecule at an undefined location

      • Examples: transposable elements

  • The integration of phage and bacterial chromosomes is an example of conservative site-specific recombination (CSSR)

    • CSSR relies on the alignment and recombination of specific, highly conserved DNA sequences

  • Once the phage has integrated into the bacterial genome, the phage is transcriptionally silent except for the cI gene

    • Continued expression of cI protein maintains lysogeny, and it keeps the cI gene active and cro repressed

  • For the phage, the evolutionary advantage of lysogeny is that its genes are safely replicated and propagated by the host bacterium

    • But if the bacterium is in danger, so is the prophage

  • If a bacterium experiences DNA damage, it initiates its own disaster plan called the SOS response

    • The bacterium uses SOS to activate DNA repair mechanisms, including homologous recombination

    • To permit homologous recombination, bacteria undergoing SOS produce the active form of the protein RecA

  • If the bacterium contains a lambda prophage, the prophage uses the cell’s SOS response as a warning signal to initiate excision and try to escape the potentially dying cell by entering its lytic cycle

    • The phage’s cI protein has evolved to undergo autocleavage if it comes into contact with activated RecA

    • Thus, the bacterial SOS response causes a rapid drop in the concentration of cI protein within the host cell

  • When the prophage is in maintenance phase:

    • There is abundant cl protein

    • The cI gene is actively transcribed (positive autoregulation)

    • The cro gene is repressed

  • During the SOS response

    • cI protein is degraded

    • Without cooperative binding to cI protein, RNA Pol cannot associate with the PRM promoter

    • The cI gene ceases to be transcribed

  • Exit from lysogeny

    • In the absence of the cI repressor protein, the strong PR and PL promoters resume transcription, including the synthesis of Cro

    • Integrase reverses its prior function, excising the prophage from the bacterial chromosome

    • The lytic cycle ensues

  • When lambda phage needs to “jump ship”, it must do so quickly

    • Therefore, regulation of cI transcription is finely tuned to produce a concentration of cI protein that is optimal for the survival and propagation of the phage

  • Optimal Concentration

    • Excess cI protein

      • Prophage will be slow to exit from lysogeny, and may be destroyed if the host cell dies rapidly

      • Negative regulation

    • Insufficient cI protein

      • Prophage may exit from lysogeny even though the host cell is healthy

      • Positive regulation

  • Even if the concentration of cI protein is low, it can bind at the high affinity OR1 operator, which can recruit another cI dimer at OR2 through cooperative binding

  • Binding of cI to OR2 is followed by the recruitment of RNAP to the PRM promoter, increasing cI synthesis

  • All cellular proteins have some rate of turnover (they are eventually broken down into amino acids)

    • But if a prophage is synthesizing cl protein faster than cl is being turned over, the concentration of cl will rise until the optimal concentration is reached

  • If cl protein can occupy the OR3 operator, it would block the PRM promoter and repress its own transcription

    • OR3 is a low affinity binding site

    • The cl protein at OR2 is already part of a tetramer, and cannot bind cooperatively with protein at OR3

  • The PL promoter has a set of three similar operator sequences that can also bind cl protein

  • As the concentration of cl protein increases, the probability that it occupies each of its binding sites also increases

  • If the concentration of cl protein is high enough, Operators 1 and 2 will be simultaneously occupied by protein on both the right and left promoters

    • When this occurs the cl proteins can form an octamer, looping the DNA

    • This looping physically aligns with the OR3 and OL3 operators allowing cl dimers to bind OR3 and Ol3 and also bind cooperatively with each other to form a tetramer

  • When the concentration of cl protein is high enough that all six operators are simultaneously bound by cl protein, the PRM promoter is blocked, and RNA Pol can no longer transcribe cl

    • As a result, the concentration of cl protein drops

  • Mini Study Guide

    • Be able to predict what perturbations of the regulatory system would do

    • Know the phases of the lysogeny cycle

    • Know how the phage detects whether the cell is healthy or dying

      • In each case, what is the optimal course of action for the phage?

    • What is conserved site-specific recombination?

    • Explain how phage exist lysogeny and return to the lytic cycle

    • Understand how levels of cl protein undergo positive autoregulation and negative autoregulation

  • I-Clicker Questions

    • Be able to draw out the phage lambda regulatory region

    • What is the first gene that must be expressed for lysogeny to be possible?

      • cll

    • What E. coli protease is capable of degrading cll?

      • FtsH

    • When are FtsH concentrations high?

      • when the cell is actively dividing

    • Why does the phage choose lysis in a healthy and dividing cell?

      • There are likely more cells nearby to infect so the phage can maximize reproduction

    • When cll is degraded by FtsH, which transcription factor gains the upper hand?

      • cro

    • What happens when multiple phages infect a single E. coli cell?

      • FtsH « cll, lysogeny

    • Typical Test Question: A lambda bacteriophage experiences a frameshift mutation in the protein-coding region of its cll gene. What effect would you expect this to have on the phage’s phenotype?

      • Phage would be able to commit to the lytic pathway, but not to the lysogenic pathway

    • Typical test question: Which of the following would not favor the commitment of a bacteriophage to lysogeny?

      • There is sustained expression of the cro gene from the PR promoter.

    • Where does the phage genome integrate into the bacterial genome?

      • At a specific sequence recognized by the integrase enzyme

    • Which transcription factor is called the lambda repressor?

      • cl

    • Why is cl called the lambda repressor

      • It stops the phage from enter lysis

      • It keeps the phage genome integrated into the bacterial genome

      • It prevents exit from lysogeny

      • It prevents death of the cell

    • Which protein is detected by the phage as a sign that the cell is in trouble?

      • RecA

    • How does cl respond to the presence of RecA?

      • Autocleavage

    • Typical Test Question: In order to re-enter lysis quickly, the phage must maintain an optimal concentration of cl. Which process does not contribute to maintaining the optimal concentration?

      • Autocleavage

      • DOES INVOLVE:

        • Negative autoregulation

        • Positive regulation

        • Coopertive binding

    • How many operators can cl bind to?

      • 6 operators

    • Which operators are bound when cl forms an octamer?

      • OR1, OR2, OL1, and OL2

    • Which operators must be occupied by cl to inhibit expression of cl?

      • OR3 and OR1

    • Typical Test Question: During DNA replication in E. coli, a lambda prophage undergoes mutation of its OR3 operator such that proteins can no longer bind there. However, the -10 and -35 sequences of the PRM promoter are still intact and properly spaced. What effect will this mutation have on the prophage?

      • The cl gene will no longer show negative autoregulation