Molecular Chapter 27
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A virus that infects bacteria
Define lytic infection.
The process by which a phage infects, reproduces w/in, then kills its host
Define lysogenic infection.
The process by which a phage infects bacteria and inserts its genome into the host genome
The latent form of the phage genome w/in bacteria
Define temperate phage.
Phages that follow a lysogenic pathway
The process by which a prophage is freed form the restriction of lysogeny
The process by which prophage is incorporated into the bacterial genome
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
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
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
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
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
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
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
Define virulent mutation.
A mutation that prevents the repressor from binding the operators and sending the virus into the lytic pathway
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
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
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
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
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
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
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
Compare and contrast the events needed to establish lysogeny vs lytic replication cycles in phage lambda.
- 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
- 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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