ATF2

Usage and Biology

The cyclic AMP-dependent transcription factor ATF2 is a member of the basic leucine zipper domain (bZIP) DNA-binding protein family and is expressed by nearly all human cells. (Miller et al., 2010) In these cells, ATF2 is phosphorylated by stress-activated protein kinase (SAPK) p38 and C-Jun N-terminal kinase (JNK) at amino acids Thr69 and Thr71 in response to a specific stimulus. (Livingstone et al., 1995) This phosphorylation results in the formation of dimer structures that efficiently bind to the specific DNA consensus sequence 5′-TGACGTCA-3′ (Lin et al., 1988) in promoter regions of target genes. A short DNA fragment containing this consensus sequence element is called a cAMP response element (CRE). (Lin et al., 1988) This binding stimulates CRE-dependent transcription, thereby activating gene expression of genes involved in cell growth, stress responses, and apoptosis. (Kawasaki et al., 2000, Miller et al., 2010) An interesting discovery by Miller and colleagues in 2010 shows that ATF2 is phosphorylated by the bacterial serine/threonine kinase PknB, specifically at Thr73. This phosphorylation activates ATF2 and leads to transcription of genes.

We used ATF2 as a transcription factor in our cell-based beta-lactam sensor, which in contrast to CcpA (K5317014) or GraR (K5317015) is derived from a eukaryotic background, to translate the PknB-detected signal into reporter gene expression.

Cloning

Theoretical Part Design

The ATF2 gene was synthesized, and the gene sequence was explicity chose from its cDNA to exclude intons and shorten the gene sequence inserted into the final plasmid for characterization (K5317021).

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 21
Illegal EcoRI site found at 283
Illegal XbaI site found at 324
Illegal PstI site found at 731
Illegal PstI site found at 1180
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 21
Illegal EcoRI site found at 283
Illegal PstI site found at 731
Illegal PstI site found at 1180
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 21
Illegal EcoRI site found at 283
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 21
Illegal EcoRI site found at 283
Illegal XbaI site found at 324
Illegal PstI site found at 731
Illegal PstI site found at 1180
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 21
Illegal EcoRI site found at 283
Illegal XbaI site found at 324
Illegal PstI site found at 731
Illegal PstI site found at 1180
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 1031

Characterization

The functionality of ATF2 in our cell-based sensor was assessed by analysing its general expression after transfection in HEK293T cells and assessing its localization by linking ATF2 with the reporter gene mRuby2 (K5317001). For results please visit the registry entry K5317021.

References

Lin, Y. S., & Green, M. R. (1988). Interaction of a common cellular transcription factor, ATF, with regulatory elements in both E1a- and cyclic AMP-inducible promoters. Proceedings of the National Academy of Sciences of the United States of America, 85(10), 3396–3400. https://doi.org/10.1073/pnas.85.10.3396

Livingstone, C., Patel, G., & Jones, N. (1995). ATF-2 contains a phosphorylation-dependent transcriptional activation domain. The EMBO journal, 14(8), 1785–1797. https://doi.org/10.1002/j.1460-2075.1995.tb07167.x

Miller, M., Donat, S., Rakette, S., Stehle, T., Kouwen, T. R., Diks, S. H., Dreisbach, A., Reilman, E., Gronau, K., Becher, D., Peppelenbosch, M. P., van Dijl, J. M., & Ohlsen, K. (2010). Staphylococcal PknB as the first prokaryotic representative of the proline-directed kinases. PloS one, 5(2), e9057. https://doi.org/10.1371/journal.pone.0009057

Kawasaki, H., Schiltz, L., Chiu, R., Itakura, K., Taira, K., Nakatani, Y., & Yokoyama, K. K. (2000). ATF-2 has intrinsic histone acetyltransferase activity which is modulated by phosphorylation. 'Nature', 405(6783), 195–200. https://doi.org/10.1038/35012097