Bacteriophages II Lecture Notes PDF

LEARNING OUTCOMES At the end of this lecture, students will be able to: Explain replication mechanism in lambda phage Differentiate the regulatory mechanism involved in the lytic and lysogeny cycle The structure of the λ DNA in the phage capsid (a) and after circularization in the cytoplasm...

LEARNING OUTCOMES At the end of this lecture, students will be able to: Explain replication mechanism in lambda phage Differentiate the regulatory mechanism involved in the lytic and lysogeny cycle The structure of the λ DNA in the phage capsid (a) and after circularization in the cytoplasm (b). The DNA circularizes via the cos site.  CONTROL OF EXPRESSION IN BACTERIOPHAGE LAMBDA PL for promoter leftward PR for promoter rightward PRE for promoter for repressor establishment PRM for promoter for repressor maintenance PI for promoter for integration PR’ for secondary rightward promoter The location of the six major promoters on the λ genome and the direction in which they specify mRNA production.  WHAT HAPPENS TO THE LAMBDA GENOME AFTER IT STABILIZED? E. coli RNA polymerase interacts with PL to give rise to a short mRNA transcript that is translated into the N protein. E. coli RNA polymerase interacts with PR to give rise to a short mRNA transcript that is translated into the Cro protein. The N protein anti-terminates by binding to RNA polymerase after a specific base pair sequence, located upstream of the transcriptional termination site, has been transcribed into mRNA (nut). Other E. coli proteins contribute to anti-termination. These proteins have been named Nus, for N utilization substance. The first transcription and translation events that take place on the l genome after infection. (a) Transcription from PL leads to the production of N protein. Transcription from PR leads to Cro protein. (b) N is an anti- terminator that allows RNA polymerase to read through the tL and tR1 terminators. From PL, N and CIII proteins will be produced. From PR, Cro, CII, O, P, and Q proteins will be produced. (c) N binds to the nutL site on the DNA. In conjunction with four bacterial proteins, NusA, NusB, NusD, and NusE, N allows RNApolymerase to read through the terminator t. LAMBDA AND THE LYTIC–LYSOGENIC DECISION The decision depends on the amounts of two phage-encoded proteins called CI (pronounced C- one) and Cro, and their binding to their promoter control regions. When CI is bound, the expression of the lytic genes is repressed and the phage follows the lysogenic pathway. For this reason, CI is also known as CI repressor or lambda repressor. The expression and binding of Cro leads to lytic development.  Cro is made from PR and CI is made from either PRE or PRM. Both Cro and CI bind to the same DNA sequences called operators. Lambda contains two operators that bind Cro and CI. One, called OR, overlaps the PRM and PR promoters. OR is a major player in the lytic–lysogenic decision, while OL is not part of the decision. OR is composed of three 17 base pair sequences called OR1, OR2, and OR3. CI repressor binds to OR1 10 times better than it binds to OR2 or OR3.  At increasing concentrations of CI, it will bind to OR2 and eventually to OR3. When CI is bound to OR, it stimulates the PRM promoter and the production of CI repressor and inhibits the PR promoter and the production of Cro, leading to lysogeny. Cro also binds to OR1, OR2, and OR3 but in the reverse order from CI repressor.  Cro binds to OR3 first, then OR2, and finally at high concentrations to OR1. When Cro is bound to OR, it inhibits the PRM promoter and the production of CI, leading to lytic growth. This is the basis for either lytic or lysogenic growth.  CI and Cro are the proteins responsible for the two developmental fates of lambda. (a) CI leads to lysogeny and Cro leads to lytic growth. (b) Both CI and Cro bind to two operator regions, OR and OL. OR overlaps with both PR and PRM. OL overlaps with PL. (c) OR is required for the switch between developmental pathways. It is composed of three 17 base pair sequences called OR1, OR2, and OR3. They are similar in sequence but not identical. (d) CI binds to OR1 first then OR2. It will bind to OR3 but only at very high concentrations. When CI binds to OR, it represses transcription from PR and activates it from PRM. CI binding to OR is actually required for PRM to be activated. CI binding leads to lysogeny. (e) Cro also binds to OR1, OR2, and OR3 but in the opposite order from CI. Cro binds to OR3 first then OR2 and at high concentrations OR1. Cro binding to OR3 inhibits PRM and leads to lytic.  HOW DOES THE PHAGE SWITCH BETWEEN THESE TWO DEVELOPMENTAL PATHWAYS? CII activates the PRE and PI promoters. This leads to the production of repressor and the integrase protein, which is also needed for lysogeny. When lambda infects a cell, transcription automatically begins from PL and PR using host proteins. Transcription from PR leads to production of both the Cro and CII proteins. If CII is active it will lead to production of CI, Integrase and lysogeny. If CII is inactive then Cro will repress PRM, preventing expression of CI and leading to lytic growth.  CII protein is inherently unstable. Degraded by the bacterial-encoded HflA protease. When cells are actively growing in nutrient-rich conditions, the amount of HflA in the cell is high, leading to degradation of CII and lytic growth. When cell are growing slowly, HflA levels are low, leading to stabilization of CII, production of CI, and lysogeny. The CII protein is the major player in the switch between lytic and lysogenic growth. CII is unstable and rapidly degraded by the host encoded HflA protease. Inactive CII leads to lytic growth. CII can be protected by the phage encoded CIII protein. Active CII leads to lysogenic growth.  TERMS PL is the promoter responsible for transcription of the left-hand side of the lambda genome, including N and cIII. OL is a short non-coding region of the phage genome (approximately 50bp) which lies between the cI and N genes next to PL. PR is the promoter responsible for transcription of the right-hand side of the lambda genome, including cro, cII and the genes encoding the structural proteins. OR is a short non-coding region of the phage genome (approximately 50bp) which lies between the cI and cro genes next to PR. cI is transcribed from its own promoter and encodes a repressor protein of 236 amino acids which binds to OR, preventing transcription of cro but allowing transcription of cI, and to OL, preventing transcription of N and the other genes in the left-hand end of the genome. cII and cIII encode activator proteins which bind to the genome, enhancing the transcription of the cI gene.  TERMS cro encodes a 66 amino acid protein which binds to OR, blocking binding of the repressor to this site. N encodes an antiterminator protein which acts as an alternative rho factor for host cell RNA polymerase, modifying its activity and permitting extensive transcription from PL and PR. Q is an antiterminator similar to N, but only permits extended transcription from PR.