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Define phage.
A virus that infects bacteria
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Define lytic infection.
The process by which a phage infects, reproduces w/in, then kills its host
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Define lysogenic infection.
The process by which a phage infects bacteria and inserts its genome into the host genome
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Define prophage.
The latent form of the phage genome w/in bacteria
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Define temperate phage.
Phages that follow a lysogenic pathway
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Define induction.
The process by which a prophage is freed form the restriction of lysogeny
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Define integration.
The process by which prophage is incorporated into the bacterial genome
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What are the basic aspects of a lytic phage life cycle?
- 1. Infection - Phage attaches to bacterium
- 2. DNA - injected into bacterium
- 3. Early Development - Enzymes for DNA synthesis are made and replication begins
- 4. Late Development - Genomes, heads, and tails are made and DNA is packaged into assembled virus
- 5. Lysis - Cell is broken to release progeny phages
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Describe the lytic cascade of phage gene regulation.
- 1. Early - phage genes are transcribed by host RNA pol
- 2. Middle - early product causes transcription of middle genes
- 3. Late - middle produce causes transcription of late genes
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How is the lytic cascade controlled?
- 1. At transcription initiation - early genes are switched off when middle genes are transcribed
- 2. At transcription termination - early genes are expressed together with late genes
- 3. A phage can synthesize a new sigma factor to replace the host sigma factor or synthesize a new RNA pol
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Describe the general phage lambda life cycle.
- 1. Lambda has 2 immediate early genes, N and cro
- 2. pN is an antitermination factor that allow RNA pol to continue transcription of the delayed early genes
- 3. pQ is the product of a delayed early gene and is an antiterminator that allows RNA pol to transcribe the late genes
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What are the functions of the genes in the phage lambday immunity region?
- 1. cIII - Positive regulator
- 2. N - Antiterminator
- 3. cI - Repressor
- 4. cro - Antirepressor
- 5 cII - Positive regulator
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Diagram the phage lambda lysogeney maintenance circuit.
- 1. Repressor acts at the left operator and right operator to prevent transcription of the immediate early genes (N and cro)
- 2. It also acts at the promoter PRM to activate transcription by RNA pol of its own gene
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Define lambda immunity.
If a second lambda phage DNA enters a lysogenic cell, repressor protein synthesized from the resident prophage genome will immediately bind the OL and OR in the new genome, preventing it from entering the lytic cycle
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Define virulent mutation.
A mutation that prevents the repressor from binding the operators and sending the virus into the lytic pathway
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Describe the general structure of the lambda repressor protein.
- 1. N-terminal and C-terminal domains connected by a connector
- 2. The C-terminal domains form dimers and the N-terminal domains bind DNA
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What is meant by a helix-turn-helix motif?
- 1. This is the motif in the N-terminal region of the lambda repressor protein
- 2. It allows the domain to fit into successive major groves of DNA
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Describe the means by which lambda repressor interacts w/an operator sequence.
- 1. Helix-3 of each monomer lies in the wide groove on the same face of DNA and helix-2 lies across the groove
- 2. The operators recognize the aa sequence of helix-3
- 3. The N-terminal domains extend like arms around the back of the DNA strand and bind to G residues in the major groove and the phosphate backbone, contributing heavily to the binding affinity by ~1000 fold
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How is the lysogenic circuit maintained in phage lambda?
- 1. The DNA-binding region of repressor at OR2 contacts RNA pol and stabilizes its binding to PRM (Autoregulatory control)
- 2. Repressor binding at OL blocks transcription of N from PL
- 3. Repressor binding at OR blocks transcription of cro, but also is required for transcription of cI
- 4. Repressor binding to the operators therefore simultaneously blocks entry to the lytic cycle and promotes it own synthesis
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What is the role of cooperative binding in regulation of the cI gene?
- 1. Repressor dimers at OL1 and OL1 interact with dimers bound at OR1 and OR2 to form octamers
- 2. This brings OL3 into proximity to OR3 where repressors and bind and interact and restrict cI transcription
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What is the role of cII and cIII proteins in establishing lysogeny?
- 1. They are necessary for RNA pol to initiate transcription at the promoter PRE
- 2. cII acts directly at the promoter and cIII protects cII from degradation
- 3. Transcription from PRE leads to synthesis of repressor and also blocks the transcription of cro
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Why is cII protein required for PRE activation?
- 1. PRE has atypical sequences and -10 and -35
- 2. RNA pol binds the promoter only in the presence of cII
- 3. cII binds to sequences close to the -35
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Compare and contrast the events needed to establish lysogeny vs lytic replication cycles in phage lambda.
- Lysogeny
- 1. cII and cIII cause repressor synthesis to establish and also trigger inhibition of late gene transcription
- 2. Establishment of repressor turns off immediate and delayed early gene expression
- 3. Repressor turns on the maintenance circuit for its own synthesis
- 4. Lambda DNA is integrated into the bacterial genome at the final stage in establishing lysogeny
- Lytic
- 1. Cro binds tot he same operators as teh lambda repressor, but w/different affinities
- 2. When Cro binds to OR3, it prevents RNA pol from binding to PRM and blocks the maintenance of repressor promoter
- 3. When Cro binds to other operators at OR or OL, it prevents RNA pol from expressing immediate early genes, which indirectly blocks repressor establishment
- Lysogeny vs Lytic
- 1. The delayed early stage when both Cro and repressor are being expressed is common to both cycles
- 2. The critical event is whether cII causes sufficient synthesis of repressor to overcome the action of Cro
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