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Modelling

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Bacterial Light Switch!

Background

Repressors

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.

Transcription Termination and Anti-Termination

Termination

Termination can fall into one of two catagories:

• Intrinsic Termination
• Factor-dependent Termination

Intrinsic Termination can be found to occur at defined template sequences, usually a region of hyphenated inverted sequence symmetry followed by a run of T residues. Termination through intrinsic terminators is stimulated by additional factors, e.g. NusA. Termination occurs due to the stem-loop structure formed by the base-pairing of mRNA with itself caused by inverted sequence symmetry, followed by the run of T residues. The NusA protein causes the RNA-p complex to temporarily stall at the stem-loop structure, when this is followed by a poly-A tail, the RNA-DNA duplex is destabilized. This causes the RNA-p to dissociate from the DNA, thereby terminating transcription.

Factor-dependent Termination occurs due to events that are not directly related to transcription, such as the release of ribosomes from nascent transcript or DNA damage. One such host termination factor is Rho, which acts on many sites along the bacterial chromosome. MFD, is a host termination factor that is responsible for releasing RNA-p stalled at sites of UV-induced DNA lesions.

Anti-Termination

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. 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.

A more detailed explanation of these anti-termination mechanisms will be posted later on.

N-protein plasmid

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

• (Franklin et.al. 1989) NC Franklin, JH Doelling - Am Soc Microbiol "Overexpression of N antitermination proteins of bacteriophages lambda, 21, and P22: loss of N protein specificity." - Journal of bacteriology, 1989

• (Jensen 2004) Ole Nørregaard Jensen, “Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry,” Current Opinion in Chemical Biology 8, no. 1 (February 2004): 33-41.