Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Genomics:GTL Program Projects
Shewanella Federation
38
Optical Methods for Characterization of Expression Levels and Protein-Protein Interactions in Shewanella oneidensis MR-1
Natalie R. Gassman1* (ngassman@chem.ucla.edu), Xiangxu Kong1, Gopal Iyer1, Younggyu Kim1, and Shimon Weiss1,2
1University of California, Los Angeles, CA and 2University of California, Santa Barbara, CA
Biological networks are dependent on a delicate balance of cellular signaling and dynamic transcriptional response, and it has become increasingly important to unravel these networks by accurately quantifying gene expression levels and mapping protein-protein interactions. We have developed a single optical technique, Alternating Laser Excitation (ALEX), which can integrate the analysis of protein-protein interactions and gene expression levels in a sensitive and potentially high-throughput manner.
A protein-protein interaction rich system in MR-1 is transcriptional regulation. Using the sigma factor, σ24, and the DNA bending protein IHF, we are currently reconstructing an active transcription system from MR-1 and creating fluorescently labeled proteins for interaction analysis. When fluorescently labeled proteins are characterized, the ALEX method can be used to examine the mechanistic process of gene regulation by σ24 -RNA polymerase (RNAP) from the formation of open complex to transcription elongation, and the dynamics of DNA bending by IHF for transcriptional initiation can be resolved.
Additionally, we are currently expanding the capabilities of the ALEX technique for gene expression analysis. To demonstrate advances in this effort, we examined a DNA model system using two-color coincident detection. By hybridizing two spectrally distinct fluorophores to the target, we detected and quantified individual DNA molecules. Hybridized complexes were detected by colocalization of the probes to the target and specificity of the probes was determined by Forster resonance energy transfer (FRET) between the probes. With ALEX, we have detected and quantified interactions at the DNA level and are currently focusing our efforts at the mRNA level. Progress in both quantification of gene expression and protein-protein interactions will be reported.
* Presenting author
