Role of RpaA in Circadian Gene Expression in S-Elongatus
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This paper documents a proposal that is targeted at understanding the role RpaA plays in the orchestration of circadian timing in the cyanobacterium Synechococcus elongatus. The paper explains that while the genetic code of many species provides instructions for an intrinsic biologic clock with a period of ~24h, S.elongatus is the only prokaryotic model organism for circadian timing, and that the presence of a biologic clock and the timing of certain physiological processes to a particular time of the day may prove to be an extremely valuable clinical tool and have considerable medical implications. The paper hypothesizes that RpaA acts in an OmpR-like fashion and propose to test this hypothesis by performing the following specific aims: a) mutational analysis of non-conserved residues in RpaA and OmpR-type response regulators b) determine the RpaA-DNA-binding consensus site and c) identify novel protein-protein interactions in the RpaA pathway. Included are pertinent figures and illustrations. The paper concludes that, together, these three aims will elucidate the mechanism of RpaA action and will thus allow the critical assessment of proposed models for circadian timing in S.elongatus.
From the Paper:"For our DNAse I footprinting and EMSA experiments, the original E.coli cloning vectors that Takai et al. (2006) used to create their bioluminescent reporter strains will be obtained from Dr. Kondo's laboratory. Plasmid DNA will be amplified and purified according to standard methodology. Next, with a specific restriction enzyme digest the plasmid DNA will be cut and the DNA-insert will be purified from the vector DNA by gel-electrophoresis. Bacterial expression of a recombinant RpaA will be performed utilizing a pGEX-P-1-RpaA vector, a generous gift of the Kondo laboratory, according to the procedures described by Takai et. al (2006). Purification will be carried out by standard GST-affinity chromatography, followed by PreScission (GE Healthcare) protease cleavage of the affinity tag and an anion exchange chromatography step.
"To test for RpaA-DNA-binding, we will perform electrophoretic mobility shift assays (EMSA) and DNAse I footprinting assays. As first prospective interaction targets we will test the promoter sequences that have been shown to be affected by an RpaA knockout (see above). For the detection of a DNA-protein complex, the purified DNA sequences will be endlabeled using a 32P -labeled deoxynucleotide. For the EMSA experiments suitable reaction mixtures containing labeled DNA of interest, non-specific competitor DNA, and protein will be subjected to native polyacrylamide gel electrophoresis as described by Laniel et al. (2001) 34 . In parallel, we will also utilize the endlabeled DNA-sequences for DNAse I footprinting experiments to a) confirm positive EMSA hits and b) account for the fact that RpaA might not 'gel shift'. Briefly, the footprinting reaction is done in three stages: binding of the protein to the DNA, partial digestion of the protein-DNA complex by DNAse I, and separation of the digested fragments on a DNA sequencing gel 35. To ensure our assays are performed correctly, we will also analyze the well-studied DNA-binding protein HGF (hepatocyte growth factor) 36; as a negative control we will employ actin, which has no reported DNA affinity."
Sample of Sources Used:
- Aiba H, Nakasai F, Mizushima S, Mizuno T. Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, results in stimulation of its DNA-binding ability. J Biochem (Tokyo) 1989 Jul;106(1):5-7.
- Bell-Pedersen D, Cassone VM, Earnest DJ, Golden SS, Hardin PE, Thomas TL, Zoran MJ. Circadian rhythms from multiple oscillators: Lessons from diverse organisms. Nat Rev Genet 2005 Jul;6(7):544-56.
- BLAST Alignment of RpaA and OmpR-type response regulators [Internet. Behtesda, MD: National Center for Biotechnology Information. 09/27/07]. Available from: http://www.ncbi.nlm.nih.gov/BLAST/.
- Bryant D. The Synechoccus sp. PCC7002 Genome Sequencing Project [Internet]; c2007 Available from: http://www.bmb.psu.edu/faculty/bryant/lab/synechococcus7002genome/index.html#The%20Organism.
- Caslake LF, Gruber TM, Bryant DA. Expression of two alternative sigma factors of synechococcus sp. strain PCC 7002 is modulated by carbon and nitrogen stress. Microbiology 1997 Dec;143:3807-18.
Cite this Research Proposal:
Role of RpaA in Circadian Gene Expression in S-Elongatus (2010, May 16) Retrieved April 10, 2020, from https://www.academon.com/research-proposal/role-of-rpaa-in-circadian-gene-expression-in-elongatus-119658/
"Role of RpaA in Circadian Gene Expression in S-Elongatus" 16 May 2010. Web. 10 April. 2020. <https://www.academon.com/research-proposal/role-of-rpaa-in-circadian-gene-expression-in-elongatus-119658/>