Team:DTU-Denmark/Regulatory sytems

From 2010.igem.org

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Few papers describe and test the the actual needed distance from the nut-site to the termination steam loop. In vitro it is found that NusA and N is sufficient to prevent termination, and that N alone can not induce anti-termination. In the tested construct the nutL-site was placed 200nt upstream of the Lambda right terminator (Whalen et.al.1988).
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Few papers describe and test the the actual needed distance from the nut-site to the termination steam loop. In vitro it is found that NusA and N is sufficient to prevent termination, and that N alone can not induce anti-termination. In the tested construct the nutL-site was placed 200nt upstream of the Lambda right terminator (Whalen et.al.1988). There have not been investigations into the required distance from the boxB in the nut-site and to the terminator stem loop. Either is the function of the boxC site fully reported. In our construct we have 26 bp from the boxB site to the beginning of the terminator stem loop, see design of our switch for more in formation.  
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Revision as of 08:43, 26 October 2010

Welcome to the DTU iGEM wiki!


The lambda phage

The 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.

Figure 1: The two distinct development pathways of a prophage life cycle [3].
In the lytic pathway the phage uses the bacterial molecular machinery to make many viral copies for infection of other cells before lysing the host bacterium. In contrast to the lytic pathway, the phage integrates its DNA into the bacterial genome in the lysogenic pathway. The lysogenic states is very stable, which means that the prophage can be replicated along with the bacterial genome for generations.
Despite the stability of the lysogenic state, the lytic state is readily induced when the bacteria are irradiated with ultraviolet light.


The organization of the phage chromosome is shown in Figure 2.

Figure 2: Schematic representation of the genome of the bacteriophage lambda [5].


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 Gifsy2

Gifsy 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].

Figure 2: The positions of the prophage inserts into the Salmonella genome [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 organization

The 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].


Figure 4: The immunity region in Gifsy phages is illustrated [1].


Regulation of promoters and repressors

GogR 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 System

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Alpha-repressor

The 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 systems

Termination

In e. coli termination is controlled by many factors, and interaction between the DNA-sequence, the RNA-structure,and native regulatory factors. Termination can be affected, enhanced or suppressed by both native and introduced phage regulatory proteins. Termination sites can in general be divided into two categories:

  • Intrinsic termination occurs at the inverted sites followed by T residues, this forms the classical termination hairpin stem loops that interact with the NusA protein, native to e. coli, to induce termination, see figure below
  • Factor-dependent termination Is not clearly defined by sequences, but by interaction between the Rho termination protein, or other factors, and the RNA-polymerase (RNAP), or release of the RNAP at DNA damage sites
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)

Figure 2: Illustration of intrinsic termination. The RNAP pauses at the terminator site due to low binding affinity because of the poly A region. NusA binds to the RNAP and stimulate hairpin formation that cancels transcript, by weakening the contact between the RNAP and the RNA strand. (Nudler et.al.2002)


Antitermination

Lambda phage regulates it's gene by an anti-termination system. The mechanism is controlled by the N protein, other phages uses the same mechanism as P21 and P22. The mechanism of the antitermination is that the N protein binds to Box B in the nut-site in the transcribed RNA. This makes another NusA bind to the spacer region in the nut-site, and prevent the formation of the termination stem loop. N further modifies the transcribing complex to proceed faster and also suppress Rho-dependent termination. (Burmann et.al. 2010)

Figure 3: The N protein Anti-termination complex. All the regulatory factors: NusA, NusB, NusE and NusG are native to e. coli except the Lambda N protein(Burmann et.al. 2010)


The N Protein binds to the nutsite upstream of the terminator site, the nutsite consists of two conserved sequences called the boxA and boxB site, the boxB site forms a small stem loop. Nus A binds the spacer region between boxA and boxB and by changing this sequence the anti-termination is prevented. Examples of the nut left and right sites from Lambda phage can be seen in the figure below. The affinity of NusA for nutL is 50% higher than for nutR (Prasch et.al. 2009).

Figure 4: The nut-sites, the regulatory RNA sequences that binds the anti-termination protein N and the NusA factor and initiates anti-termination.(Prasch et.al. 2009)


Few papers describe and test the the actual needed distance from the nut-site to the termination steam loop. In vitro it is found that NusA and N is sufficient to prevent termination, and that N alone can not induce anti-termination. In the tested construct the nutL-site was placed 200nt upstream of the Lambda right terminator (Whalen et.al.1988). There have not been investigations into the required distance from the boxB in the nut-site and to the terminator stem loop. Either is the function of the boxC site fully reported. In our construct we have 26 bp from the boxB site to the beginning of the terminator stem loop, see design of our switch for more in formation.

Another Anti-terminator mechanism is seen for the lambda Q protein. It also functions by RNAP modification, but the Q protein binds to the DNA upstream of the Promoter. the Bound Q protein interacts with the RNAP and modifies it to faster elongation and termination resistance (Burmann et.al. 2010).

References

  • Burmann. B., Schweimer. K., Luo. X., Wahl. M., Stitt. B., Gottesmann. M., Rösch. P., " A NusE:NusG Complex Links Transcription and Translation" Science 2010.
  • Nudler. E., Gottesman. M.E., "Transcription termination and anti-termination in E.coli", Genes to Cells (2002)
  • Prasch. S., Jurk. M., Washburn. R.S., Gottesman. M.E., Wöhrl. B., Rösch. P., "Rna-binding specificity of E. coli NusA" Nucleic Acids Research 2009.
  • Whalen. W., Ghosh. B., Das. A., "NusA protein is necessary and sufficient in vitro for phage lambda N gene product to suppress a rho-independent terminator placed downstream of nutL. Proc.Natl. Acad sci. 1988