Week 5 S - Gene Regulation in Prokaryotes - Phage Lambda

Gene Control in Phage Lambda

Phage Lambda Life Cycle

• Two primary states:
  • Lysis:
    • Phage DNA replicates, and proteins are synthesized.
    • Results in the production of progeny phages and cell lysis.
  • Lysogeny:
    • Phage DNA integrates into the host chromosome.
    • A single phage protein (cI product, the lambda repressor) is produced.

Lambda Transcription

• Two major transcription units are controlled by two adjacent promoters:
  • P_L (leftward promoter).
  • P_R (rightward promoter).

Key Regulatory Elements

• N: Antiterminator protein.
• cI: Lambda repressor protein, crucial for maintaining lysogeny.
• cro: Cro repressor protein, important for the lytic cycle.
• Immunity Region: Regulatory locus containing PL/OL, PR/OR, and P_{RM}.
• tL, tR1: Transcription terminators.
• PL/OL: Leftward promoter and operator region.
• PR/OR: Rightward promoter and operator region.
• PRM: Promoter for repressor maintenance.

Repressor Function

Lysogenic Cycle

• The repressor protein maintains the lysogenic state.
• Repressor dimers bind to operator sites (e.g., OL) and prevent RNA polymerase from binding to PL, thus inhibiting transcription from P_L.
• Repressor monomers are produced from the cI gene, transcribed from P_{RM}.
• RNA polymerase binds to P{RM} and transcribes cI, but the repressor also prevents RNA polymerase from binding to PR.

Lytic Cycle

• Repressor is absent during the lytic phase.
• RNA polymerase initiates transcription at P_R, producing cro mRNA.
• In the absence of the repressor, RNA polymerase cannot initiate at P_{RM}.

Structure of PR and P{RM}

• PR has a nearby leftward promoter, P{RM}, which is the promoter for repressor maintenance.
• PR and P{RM} are overlapped by an operator, O_R.
• O_R consists of three 17 base pair (bp) sites.

Operator Regions OR & OL

• Both lambda repressor and Cro can bind to six operator sites in the OR and OL regions.
• OR includes sites O{R3}, O{R2}, and O{R1}. OL includes O{L3}, O{L2}, and O{L1}.
• Overlapping of operators and promoters occurs at PR and PL.
• The arrangement dictates the transcription of cI and cro.

Operator Site Sequences

• The six operator sites recognized by Lambda Repressor and Cro:
  • O{L1}, O{R1}, O{L2}, O{R2}, O{L3}, O{R3}

Operator Structure

• Each of the six sites is a palindrome (imperfect).
• OR and OL are closely similar.
• The consensus sequence for the half site is T9A{12}T6C{12}A9C{11}C7G9(G/C)_{5/5}.

Lambda Repressor Structure

• Composed of an N-terminal domain and a C-terminal domain.
• Monomers are in equilibrium with dimers, which bind to DNA.
• Repressor cleavage induces the lytic cycle, shifting the monomer-dimer equilibrium.
• Cleavage of monomers disturbs the equilibrium, causing dimers to dissociate.

Helix-Turn-Helix (HTH) Domains

• Two helix-turn-helix domains are required for the λ repressor to bind DNA.
• The N-terminal domain consists of 5 α-helices.
• Repressor binds DNA via two α-helices.

Repressor Binding to DNA

• The repressor primarily contacts one face of the DNA duplex.
• N-terminal arms reach around the duplex to contact the opposite face.
• Lysines in the arm make G contacts in the major groove.
• ‘Armless’ mutants exhibit a 1000x reduction in DNA-binding affinity.

Cro Protein Structure

• A small protein of 66 amino acids.
• Exists as a monomer that dimerizes.
• The dimer binds to DNA.

HTH of λ Repressor and Cro

• Helix 3 determines DNA-binding specificity.
• Residues involved in positioning helix 3 use H-bonds.
• Each protein makes slightly different side-chain contacts with the operator base-pairs, resulting in sequence-specific interactions.

Repressor Binding to O_R

• Each subunit of each dimer binds one subunit of the other dimer.

RNA Polymerase Interaction

• A part of helix 2 interacts directly with RNA polymerase to promote promoter occupancy and activation of transcription.
• Repressor binding at O{R2} enhances RNA polymerase binding at P{RM}.
• Auto-activation of P{RM} when ON and auto-repression of P{RM} when OFF.

Lambda Repressor forms Octamers

• Binding of the repressor is cooperative.
• Reduces the concentration of repressor required to occupy operators by x3.
• Increases the efficiency of induction because less repressor needs to be eliminated when phage induction is necessary.

Cro Protein Binding to O_R

• Cro binds to O_R sites, competing with the lambda repressor.

Lysogenic/Lytic Trigger

• λ repressor favors lysogeny.
• Cro favors lysis.
• Competition between Cro and λ repressor determines the life cycle.
• Lysogeny: λ repressor occupies OL and OR. P{RM} is ON, while PL and P_R are OFF.
• Lytic phase: Cro replaces λ repressor at OL and OR. PL and PR are ON, while P_{RM} is OFF.

Conversion of Lysogenic to Lytic State

• DNA damage triggers RecA-mediated cleavage of the λ repressor.
• Cleaved λ repressor is inactive.
• The SOS response is induced, leading to LexA repressor cleavage.
• Cleaved LexA is also inactive.
• Induction of lysogen occurs due to DNA damage, such as UV light exposure.

Summary

• Cooperative binding to DNA via oligomer formation increases binding affinity.
• Transcription factors can act as both repressors and activators.
• Transcription factor concentration is a critical factor.
• Control can be exerted post-transcriptionally, e.g., by proteolysis.
• DNA-binding affinity is sequence-dependent.
• Regulators can auto-regulate their own expression (auto-control).