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Lambda Phage Lysogeny: Site-Specific Recombination and Gene Regulation, Cheat Sheet of Molecular biology

Maharaja Sayajirao University of BarodaMolecular biology

An in-depth exploration of the lysogenic cycle of the lambda phage, focusing on prophage integration, gene regulation, and superinfection immunity. Key topics include attp site integration, homologous recombination, int and ihf proteins, and the roles of ci, cii, and cro. The document also covers the regulation of ci repressor levels and the steps in lysogeny.

What you will learn

  • How does homologous recombination facilitate lambda phage integration?
  • How does the regulation of CI repressor levels impact the lysogenic cycle?
  • What are the functions of the CI, CII, and Cro proteins in lambda phage lysogeny?

Typology: Cheat Sheet

2021/2022

Uploaded on 12/24/2022

Altayih
Altayih 🇮🇳

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Lysogenic cycle of lambda.
Prophage integration.
Lambda integration happens at a special site att'.
It is between gal and bio in bacteria - BOB'.
In phage - POP'.
Integration phenomena - Homologous recombination. (Holliday junction)
The phenomena requires phage protein Int and bacterial protein IHF (Integration Host Factor).
Both Int and IHF binds to attP' and form intasome.
Intasome is a DNA-protein-complex designed forsite-specific recombinationof the phage and host DNA.
Induction and aberrant excision (specialized transduction).
Induction and excision is carried out by Int and Xis genes.
Excision error at a very low frequency1 cell per 106 to 107 cells.
Gene product and functions.
CI - Repressor. Inhibit transcription from pL and pR.
CII - Transcriptional activator of CI and int.
CIII - Stabilizes CII.
Cro - Protein inhibitor of CI protein synthesis.
Important synthesis.
pRE - Promoter of repressor establishment. Activated by CII.
pRM - Promoter of repressor maintenance. Activated by CI.
pI - Promoter for int transcription. Activated by CII.
Superinfection immunity.
CI is produced in lysogeny continuously.
We are also ware that CI binds to oL and oR and prevents transcription from pL and pR.
The incoming phage also receives the same treatment and all transcription is stopped in it. (Because the CI produced by lysogen also binds to operators of this superinfecting phage.)
In subsequent cell division, the superinfecting phage DNA is diluted out.
Lysogeny.
When lysogenization occurs CII is synthesized to activate pRE.
CI is made from pRE and it activates pRM.
When CI is in high concentration, CII is repressed.
Since it stops transcription from pR.
Since CII is repressed, transcription from pRE stops.
CI inhibits transcription from pL and pR by binding to oL and oR.
Since CI is synthesized continuously, this keeps the phage in lysogeny indefinitely.
Immediate early transcripts.
Transcription starts from pL, pR and pR' and forms immediate early transcripts.
These express N and Cro genes and a short inactive transcript.
Crobinds toOR3, preventing access to thepRMpromoter, preventing expression of theCIgene.
Regulation of levels of CI repressor.
High and low synthesis of repressor is achieved by existence of two promoters.
pRE - Functions in infected cell and requires CII.
pRM - Functions in lysogen and is regulated by repressor itself.
Steps in lysogeny.
1. Transcription from pL and pR.
2. CII protein is translated from pR transcript.
3. CII enables RNA polymerase to transcribe from pRE promoter. The CI repressor is made from pRE transcript.
4. CI binds to oL1 and oR1 and inhibits transcription from pL and pR. The CII protein is unstable, so transcription from pRE stops.
pf3

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Lysogenic cycle of lambda.

Prophage integration.

Lambda integration happens at a special site att'. It is between gal and bio in bacteria - BOB'. In phage - POP'. Integration phenomena - Homologous recombination. (Holliday junction) The phenomena requires phage protein Int and bacterial protein IHF (Integration Host Factor). Both Int and IHF binds to attP' and form intasome. Intasome is a DNA-protein-complex designed for site-specific recombination of the phage and host DNA.

Induction and aberrant excision (specialized transduction).

Induction and excision is carried out by Int and Xis genes.

Excision error at a very low frequency 1 cell per 10^6 to 10^7 cells. Gene product and functions. CI - Repressor. Inhibit transcription from pL and pR. CII - Transcriptional activator of CI and int. CIII - Stabilizes CII. Cro - Protein inhibitor of CI protein synthesis. Important synthesis. pRE - Promoter of repressor establishment. Activated by CII. pRM - Promoter of repressor maintenance. Activated by CI. pI - Promoter for int transcription. Activated by CII.

Superinfection immunity.

CI is produced in lysogeny continuously. We are also ware that CI binds to oL and oR and prevents transcription from pL and pR. The incoming phage also receives the same treatment and all transcription is stopped in it. (Because the CI produced by lysogen also binds to operators of this superinfecting phage.) In subsequent cell division, the superinfecting phage DNA is diluted out.

Lysogeny.

When lysogenization occurs CII is synthesized to activate pRE. CI is made from pRE and it activates pRM. When CI is in high concentration, CII is repressed. Since it stops transcription from pR. Since CII is repressed, transcription from pRE stops. CI inhibits transcription from pL and pR by binding to oL and oR. Since CI is synthesized continuously, this keeps the phage in lysogeny indefinitely.

Immediate early transcripts.

Transcription starts from pL, pR and pR' and forms immediate early transcripts. These express N and Cro genes and a short inactive transcript. Cro binds to OR3, preventing access to the pRM promoter, preventing expression of the CI gene.

Regulation of levels of CI repressor.

High and low synthesis of repressor is achieved by existence of two promoters. pRE - Functions in infected cell and requires CII. pRM - Functions in lysogen and is regulated by repressor itself.

Steps in lysogeny.

  1. Transcription from pL and pR.
  2. CII protein is translated from pR transcript.
  3. CII enables RNA polymerase to transcribe from pRE promoter. The CI repressor is made from pRE transcript.
  4. CI binds to oL1 and oR1 and inhibits transcription from pL and pR. The CII protein is unstable, so transcription from pRE stops.
  1. Occupation from oR1 and oR2 causes pRM to be activated. cI is continuously synthesized from this promoter. Lower rate of formation of CI from pRE.
  2. CI repressor continues to accumulate, do oL1 and oR2 become occupies and repression from pL and pR is complete.
  3. The concentration of CI gets so high that it binds to oR3. This inhibits pRM and that stops the synthesis of CI. The activity of pRM is turned on and off to accommodate mild fluctuations to repressor concentrations.
  4. Finally int protein integrates the phage DNA into chromosome.

Operator binding by CI.

oL is divided into three regions: oL1, oL2 and oL3. oR is divided into three regions: oR1, oR2 and oR3. The relative affinities of each isolated region is different. oL1>oL2>ol3. oR1>oR2>oR3. oL1/oR1 and oL2/oR2 binding is cooperative. oL3 and oR3 are non cooperative.

Transcriptional activator CII.

The stabilized CII acts to promote transcription from the pRE, pI and pAntiq promoters. pAntiq promoter produces antisense mRNA to the Q gene message of the PR promoter transcript, thereby switching off Q production. No Q results in no extension of the pR' promoter's reading frame, so no lytic or structural proteins are made. The pRE promoter produces antisense mRNA to the cro section of the PR promoter transcript, turning down cro production, and has a transcript of the CI gene. The Pi promoter expresses the int gene, resulting in high concentrations of int protein. This int protein integrates the phage DNA into the host chromosome.

Induction.

Any situation where a lysogen undergoes DNA damage or the SOS response of the host is otherwise stimulated leads to induction. RecA detects DNA damage and becomes activated. It is now RecA, a highly specific co-protease. Normally RecA binds LexA (a transcription repressor), activating LexA auto-protease activity, which destroys LexA repressor, allowing production of DNA repair proteins. In lysogenic cells, this response is hijacked, and RecA* stimulates CI autocleavage. This is because CI mimics the structure of LexA at the autocleavage site. Cleaved CI can no longer dimerize, and loses its affinity for DNA binding.