Team:DTU-Denmark/Regulatory sytems
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Termination can be affected by the phage anti-terminator factors as the N-protein binding to an RNA site upstream of the terminator or the Q-protein that binds the DNA directely.(Nudler et.al. 2002) | Termination can be affected by the phage anti-terminator factors as the N-protein binding to an RNA site upstream of the terminator or the Q-protein that binds the DNA directely.(Nudler et.al. 2002) | ||
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+ | <caption align="bottom"><p align="justify"><b>Figure 7</b>: The immunity region in Gifsy phages is illustrated [1].</p></caption> | ||
+ | <tr><td><img src="https://static.igem.org/mediawiki/2010/4/41/Termination_nusa.png" width="400px"></td></tr> | ||
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<h3> Antitermination </h3> | <h3> Antitermination </h3> |
Revision as of 10:16, 25 October 2010
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The lambda phageThe temperate bacteriophage lambda, E. coli, is the best-studied phage with regard to phage structure and regulation. Temperate phages are able to choose between the lytic cycle and the lysogenic cycle as described in Figure 1. The organization of the phage chromosome is shown in Figure 2.
The rightward promoter/operator region in lambda prophages forms a switch with the two genes cro and cI under mutually exclusive expression in the lambdoid phage. This regulatory region consists of the promoters pRM and pR and the three sub-operator sites, OR1, OR2 and OR3, in-between the promoter. The interesting feature about these two promoters is that they are orientated in a back-to-back fashion and are also called divergent or bidirectional promoters. The sub-operator sites are shared regulatory regions that influence the expression of two oppositely oriented genes. This type of promoter arrangement is common in prokaryotes and is also found in humans and other higher species. The pR promoter is very strong and has greater similarity to the promoter consensus sequence than the very weak pRM promoter [6]. The cI gene encodes cI, the lambda repressor protein. The presence of this protein stabilizes the lysogenic state and causes immunity to superinfections by other lambda phages. cI has a dual function by acting as repressor and activator. It represses transcription from the pR promoter while up-regulating its own expression from the pRM promoter. A dimer is formed by the cI repressor and binds to DNA in the helix-turn-helix binding motif. The cI repressor binds to all three operator sites in the order OR1 = OR2 > OR3, because of its different intrinsic affinities for the operator sites. The repressor binds with highest affinity to OR1 and that stimulates binding of more cI to OR2 by a mechanism called positive cooperative binding. Binding to OR1 and OR2 blocks binding of RNA polymerase to the pR promoter, so switching to the lytic cycle is prevented. At high repressor concentrations cI down-regulates its own expression by binding to OR3, so all three operator sites are occupied and expression from the pRM promoter is limited i.e cI, while the expression from cro genes still is inhibited. It is important to understand, that regulation of the switch is solely dependent on repressor concentrations and not by other regulatory proteins e.g. anti-repressors. The Cro protein does not bind with positive cooperation to the three operator sites whilst cI does. The result is that cI and the lysogenic state is more stable, because it can outcompete the cro protein. The lytic pathway of lambda is induced by the SOS response after DNA damage in E. coli by e.g. UV light. This is achieved when the repressor protein cI is cleaved by a protein expressed during SOS response, RecA [4]. The Gifsy phages: Gifsy1 and Gifsy2Gifsy 1 and Gifsy 2 are temperate phages present in the vast majority of Salmonella enterica serovar Typhimurium strains; the genomic positioning of the prophages is illustrated in Figure 2. This strain of pathogenic bacteria infects a broad spectrum of animal species, from reptiles to mammals [1].
Similar to the lambda phage, the Gifsy phages follow either lytic cycle or lysogenic cycle after infecting S. enterica. Salmonella strains harbor different subsets of prophages and therefore have differences in prophage distribution, lysogeny thereby contributes to the genetic diversity of Salmonella genomes. Since phages are able to switch between these two different developmental states, they are a very interesting example of a natural genetic switch and this is why we choose to use the key regulatory elements from Gifsy-1 and Gifsy-2 prophages to construct our own bistable switch in E.coli [1]. Chromosomal organizationThe overall gene organization of Gifsy-1 and Gifsy-2 prophages is typical of the lambdoid phage family that is illustrated in Figure 2 [1]. The regulation region of the lambda phage, also called immunity region, is of particular interest to us. This region includes the three promoters pR, pRM and pL, the left- and rightward operator and the cro and cI gene responsible for controlling the switch between lysogenic and lytic growth. The Gifsy chromosome and its immunity region are organized in a very similar way to that of the lambda phage as illustrated by Figure 4 [1].
Regulation of promoters and repressorsGogR and GtgR are repressor proteins found in Gifsy-1 and Gifsy-2, respectively. These repressor proteins are analogous to the lambda repressor protein, cI, previously described. The Gifsy repressors (136 aa) are much smaller than the lambda repressor cI (237 aa) and lack the typical cleavage motif [1]. The mechanism by which the repressors GogR and GtgR regulate the Gifsy promoters, pR and pRM is analogous to that of cI in the lambda phage with the exception of the mode of lytic induction. As previously mentioned, GogR and GtgR lack the cI cleavage site and are inactivated by binding of small anti-repressor proteins, called AntO and AntT. The Gifsy genes encoding these proteins are located outside the immunity region and are under the direct control of the LexA protein. This protein is the major regulator of the SOS regulon and this regulon is activated by the cleavage of LexA by RecA. The interesting point is that Gifsy and lambda prophage regulation is an integral part of the SOS response i.e. once the SOS response is triggered, the lytic pathway is also induced [1,7]. Phage Repressor SystemMaya Lisa anja Alpha-repressorThe C1-repressor is responsible for repressing transcription of the lytic genes, thereby maintaining the stable lysogenic state. The induction of the lytic state is caused by activated RecA, which stimulates the self-cleavage of the C1-repressor. We will be using the C1-repressor in our system. Anti-Termination systemTerminationIn e. coli termination is controlled by many factors, and interaction between the DNA-sequence and structure, the structure of the transript, native regulatory facotrs. Termination can be affected, enhanced or supressed by both native and introduced phage regulatory proteins. Termination sites can in general be divided into two categories:
AntiterminationINTRO ANTITERMINATION N-PROTEIN The N-protein is able to suppress transcription termination at both factor-dependent and factor-independent termination sites. N anti-termination is strongly stimulated by the NusA protein. Unlike the N-protein, the Q-protein specifically binds to a DNA sequence immediately upstream of the pR´ promoter. N-LAMBDE FACTOR DEPENDENT: Anti-Termination is the process by which the termination of gene transcription is prevented. Such control of gene transcription can be found in the phage Lambda system. The mechanism is controlled by proteins, such as the lambda N or lambda Q-proteins. The expression of early genes and late genes are both regulated by the anti-termination mechanism, controlled by the lambda N-protein and the lambda Q-protein, respectively. N-Protein The mechanism of N-protein nut-site termination have been studied heavily the last years and the current best descriped mechanism have been done in a couple of reviews (XXXXX,XXXX,XXXX) The anti-termination function by introduction of the N-protein (or equivalent) that interacts with nusA and disrupt the termination. Of known systems can be mentioned lambda, p21, p22 FUNCTION XXXXXXXXXXXX shown in the figure below. In the known systems the nut site is placed from XXX bp to XXX bp upstream of the termination steam loop. (REFERENCES !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! IMPORTANT - read two gottersman 2010 – crystallography papers)))) figures from papers on antitermination. Few papers descripe and test the the actual needed distance from the termination steam loop. From other systems and mechanism it is known that XXX bp is needed for regulation of RNAP or DNAP. (MOGENS ABOUT REFERENCES AND SYSTEMS SEE MICRO-BIO-TEXT BOOKS_) . The N protein were isolated from salmonella genomic DNA with specific designed primers. We used the natural occurring RBS site, as a High expression of N have shown non specific anti-termination effect on a global scale on the genome. [[#References References]] nut sites EngineeringSeverel papers analyse the function of the nut-site. It has been shown that this mechanism can be manipulated in different manners and that the function can be canceled and reactivated by counter-mutations in XXXXX (REFFFFF). Futher it has been shown that the specificity of the N-proteins can be changed from lambda to P22 by only a few mutations showing a possible coevolution, or possible interactions to increase possible genomic randomization References
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