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The bacteriophage lambda is a virus that is parasitic in bacteria, attaching by its tail to the surface to the surface of an E.coli cell and injecting its chromosome into the bacterium to multiply. The lambda infected bacterium then exhibits either a lytic cycle or a lysogenic cycle. Generally after infection of an E. coli host, the lambda phage chromosome lysogenizes the host. All except one gene present in the phage is turned off, hence causing one phage chromosome, the prophage, to become as part of the host chromosomes, hence making a lysogen, a bacterium containing the prophage. The lysogen in turn is replicated and distributed to the offspring bacteria whilst part of the host chromosome. In some of the cells the various genes of the lambda phage are turned on and off following a set protocol, which synthesis the heads and tails of the new protein, therefore replicating the lambda chromosome extensively in the lytic cycle. Even though these two processes are present, environmental signals such as ultraviolet rays or ionizing radiation affect the lysogenic cycle, causing lysis and release of hundreds of newly synthesised infectious phage particles. The ultraviolet rays damages the host DNA causing activation of previously dormant, turned off genes of phage lambda, leading to a change in the cycle and activation of lytic growth followed by lysis. Following this, the bacterial cell is lysed releasing hundreds of new phage particles. Present amongst the lytic and lysogenic pathways are sets of intertwined positive and negative regulators of gene expression which act pre and post transcription. Therefore, a switch-like mechanism is present which specifies whether the lambda bacteriophage will multiply within the host cytoplasm and kill the host cell or integrate itself into the host cell DNA and replicate during bacterial division.
The lytic-lysogenic switch is the resultant of the proteins encoded by the viral genome The switch is regulated by two regulatory proteins, the CI and Cro regulators, as well as two promoters, OL and OR CI and Cro define the lysogenic and lytic states, respectively, as a bistable genetic switch. CI maintains a stable lysogenic state, whereas Cro activates the lytic cycle by indirectly lowering levels of CII which activates cI transcription, hence blocking CI expression. The two regulatory proteins cI (also known as ¬ repressor) and Cro , maintain this switch and the production of either determines the fate of the infected bacterium as increase in cI proteins promotes the lysogenic cycle whereas increase in Cro proteins promotes the lytic cycle. The regulation of the transcription of both the proteins is regulated by the cI protein itself.
The lambda repressor is a dimer and is both a positive and negative regulator of gene expression, as binding to just two operators on the lambda DNA all the genes of the phage are turned off whilst turning on its gene. It regulates the transcription of the cI protein and the Cro protein.
The cI protein is made of 236 amino acids folded into two domains, amino and carboxyl, connected by 40 amino acids. They form dimers due to contacts between the carboxyl and amino domains causing associations. The amino domains present in the proteins are used to bind to the DNA. The action of the repressor protein is entwined with the attachment of the repressor dimer to the OR. OR is subdivided into three adjacent sites, OR1, OR2 and OR 3, hence forming the right operator of the phage. The cI protein plays a role in both negative and positive control. The repressor binds to the OR2, in turn turning off the cro gene, preventing binding of the RNA polymerase to PR , the right promoter. The repressor partly covers the DNA vital for polymerase binding. Hence, as the repressor binds to the OR2, the RNA polymerase is unable to gain access to the recognition sequences for the promoter.
The lambda repressor also exhibits positive control, in which case it still binds to the OR 2 but aids RNA polymerase binding and initiating transcription at PRM ,which is the promoter regulating cI transcription. During negative control the repressor switches off its own genes, however in positive control it does the opposite and only the phage genes are on, which increase transcription of its own genes,
Therefore, binding of a cI dimer to OR1 enhances binding of a second cI dimer to OR2, but not the affinity between cI and OR3. This leads to frequent occupying of the OR1 and OR2 by cI, in the presence of which only cI gene would be transcribed. However, at high concentration of cI, transcriptions of both genes are repressed.
When the host DNA is damaged (e.g., under UV irradiation), the cI protein may be cleaved by certain protease promoted by the RecA protein.Â Cleaved cI proteins cannot bind to the operators.Â Thus, the Cro proteins can be produced to transform the ¬ phage into the lytic cycle
The second regulatory protein is Cro, which is made of 66 amino acids folded into a single domain with high affinity of Cro monomers. Hence the protein is present as dimers, which bind to the three operator sites OR1, OR2 and OR 3 with different binding affinity, present in the right operator. Cro plays an active role in switching lysogenic cells to the lytic state following induction. The role and action of Cro is less complex than of the lambda repressor as it only conducts negative regulation. The dimmers bind non cooperatively to the three operator sites following its order of affinity, OR3> OR2= OR1. Thus Cro ensures the maintenance circuit for lysogeny does not come into play. Hence, following binding to the OR3, RNA polymerase binding to PRM is hindered and synthesis of repressor is inhibited. This in turn prevents the production of early functions including Cro Following this the switch is activated causing lytic growth to follow. Following PR functioning and Cro protein transcription, the Cro genes are produced the products of which are vital in early lytic growth. The amount of cro produced is maintained until saturation of the OR 1 and OR 2, which prevents polymerase binding to the PR, hence finally causing repressor synthesiss being turned off, turning off expression of own genes and other lytic genes.
The critical influence over the switch is the CII protein, which activates and coordinates transcription from three promoters pI, pRE and pAQ, which lay dormant until the presence of sufficient CII. If this protein is active, synthesis of the repressor via the promoter occurs, hence providing the repressor use of the operators. In the inactive state, the repressor production drops, allowing Cro to bind to the operators. Therefore, the amount of the CII proteins affects the outcome of the lytic or lysogenic cycle dilemma.
Obtaining a stable lambda lysogenic response with CII, specifies the need for coordination. Activation of this switch by CII, prevents the lambda phage from following the default, lytic pathway. Bacterial proteases such as HflB (FtsH) which binds to the C-terminal part of CII, causing its rapid decay, hence allow maintenance of the levels of CII. The CIII protein is also plays an indirectly, yet important role to establish lysogeny. CIII inhibits the bacterial protease HflB which provides a good means to maintain amounts of CII, which also allows the CII protein to accumulate for its action with the promoters.
Hence the preliminary event that establishes lysogeny is repressor binding at OL1 and OR1 followed by co-operative binding at OL 2 and OR2, in turn shutting off the synthesis of Cro and instead synthesising repressor via PRM. The lytic cycle on the other hand is initiated by the binding of Cro at the OR3, forcing cro to bind to OR1 or OR 2 which in turn would turn down gene expression.
The regulatory proteins Cro and cI have a helix-turn-helix motif, the three dimensional models of which show that these polypeptides share an alpha-helix-beta-turn-alpha-helix motif which is involved in specific protein-DNA interactions. These proteins are present as dimers which interact with specific DNA sequences via binding of an -helical polypeptide domain with the major groove of a symmetrically-oriented recognition site spanning one turn of a B-DNA helix.
The regulatory sites 0L and 0R including the sub dividends have dyad symmetry, and are the sites, binding at which ensures either lytic cycle or lysogenic cycle. These two proteins, repressor and Cro, bind to the same three operator sites, however play opposing roles in the switch mechanism.
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