Part:BBa_K801033

LexA DNA binding protein

LexA DNA binding protein, improved version of BBa_K105005 to allow N- and C-terminal protein fusions using RFC25 pre- and suffix.

LexA is a prokaryotic transcription factor which can be used also in eukaryotic systems to binds selectively to LexA operator sequence, which can be fused to the TATA boxes of a minimal eukaryotic promoter. If used in eukaryotic systems it is nescessary to fuse a nuclear localization signal (BBa_K801030, SV40 nuclear localization sequence) to this part to allow nuclear translocation. Of course there is no NLS nescessary in procaryotic systems.

If you want to fuse a protein domain to the N-terminus of this part and also want to ensure independent folding of protein domains you might want to use a protein linker in the N-terminus of this part. A composite parts already exists in which a 20 amino acids long linker is fused to the N-terminus of this part (BBa_K801036).

Background

Principle of light-dependent switching of gene-expression.

This system bases on the yeast two-hybrid system which was originally created for exploring protein-protein interactions. One candidate of a potential protein-interaction pair is fused to the DNA-binding domain of a transcription factor and the other candidate to the activation domain of a transcription factor. If the proteins candidates are really physically interacting with each other, this event will starts the transcription of downstream reporter genes, e. g. LacZ or an auxotrophic marker.

In contrast to the GAL4 based light-switchable promoter system there is no need for KO of GAL4/GAL80 genes in yeast with a LexA based light-switchable promoter system. The difference is that we use LexA, a prokaryotic DNA binding protein, for the DNA binding part of our light-switchable promoter system, instead of GAL4DBD. LexA does not interfere with the endogenous yeast metabolism and signaling system because it only recognizes a special prokaryotic DNA sequence, the so-called LexA operator (=LexA binding site). LexA binding sites can be used upstream of a minimal promoter (=TATA box) to be utilized as a cis-acting regulatory element.
In this case the genes, which we want to control by light, have to be cloned downstream of a synthetic promoter containing a minimal promoter, preceded by multiple LexA binding sites

References

• http://www.ncbi.nlm.nih.gov/pubmed/15823535 Chen et al., 2005 Chen, M., Tao, Y., Lim, J., Shaw, A., and Chory, J. (2005). Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. Curr Biol, 15(7):637–42.
• http://www.ncbi.nlm.nih.gov/pubmed/19165330 Kikis et al., 2009 Kikis, E. A., Oka, Y., Hudson, M. E., Nagatani, A., and Quail, P. H. (2009). Residues clustered in the light-sensing knot of phytochrome B are necessary for conformer-specific binding to signaling partner PIF3. PLoS Genet, 5(1):e1000352.
• http://www.ncbi.nlm.nih.gov/pubmed/19749742 Levskaya et al., 2009 Levskaya, A., Weiner, O. D., Lim, W. A., and Voigt, C. A. (2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature, 461(7266):997–1001.
• http://www.ncbi.nlm.nih.gov/pubmed/12355112 Mendelsohn, 2002 Mendelsohn, A. R. (2002). An enlightened genetic switch. Nat Biotechnol, 20(10):985–7.
• http://www.ncbi.nlm.nih.gov/pubmed/12219076 Shimizu-Sato et al., 2002 Shimizu-Sato, S., Huq, E., Tepperman, J. M., and Quail, P. H. (2002). A light-switchable gene promoter system. Nat Biotechnol, 20(10):1041–4.
• http://www.ncbi.nlm.nih.gov/pubmed/15486100 Khanna et al., 2004 Khanna, R., Huq, E., Kikis, E. A., Al-Sady, B., Lanzatella, C., and Quail, P. H. (2004). A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. Plant Cell, 16(11):3033–44.
• http://www.ncbi.nlm.nih.gov/pubmed/11553807 Gambetta and Lagarias, 2001 Gambetta, G. A. and Lagarias, J. C. (2001). Genetic engineering of phytochrome biosynthesis in bacteria. Proc Natl Acad Sci U S A, 98(19):10566–71.
• http://www.ncbi.nlm.nih.gov/pubmed/10466729 Ni et al., 1999 Ni, M., Tepperman, J. M., and Quail, P. H. (1999). Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. Nature, 400(6746):781–4.
• http://www.ncbi.nlm.nih.gov/pubmed/12734586 Van Criekinge and Beyaert, 1999 Van Criekinge, W. and Beyaert, R. (1999). Yeast two-hybrid: State of the art. Biol Proced Online, 2:1–38.
• http://www.ncbi.nlm.nih.gov/pubmed/3891738 Wertman and Mount, 1985 Wertman, K. F. and Mount, D. W. (1985). Nucleotide sequence binding specificity of the LexA repressor of Escherichia coli K-12. J Bacteriol, 163(1):376–84.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 534
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]