Revision as of 21:16, 1 October 2024

3xCre3xAP1-miniCMV Promoter

Usage and Biology

To be able to receive and detect the signal sent by PknB kinase activity/ATF2 phosphorylation and activation, we have developed an ATF2-responsive promoter. Based on observations made by Miller and colleagues (2010) showing similar kinase mechanisms between the prokaryotic PknB and eukaryotic MAPK towards ATF2, we generated a synthetic ATF2-responsive promoter construct with three Cre and three AP1 binding sites as well as a miniCMV promoter sequence.

The ATF2 transcription factor belongs to the ATF/CREB family and regulates genes involved in cell growth, stress responses, and apoptosis (Kirsch et al., 2020). Activated ATF2 binds to the cAMP-responsive element (CRE) with the consensus sequence 5'-GTGACGT[AC][AG]-3' (Miller et al., 2010; Hai et al.,1989). Additionally, ATF2 can form homo- or heterodimers together with members of its own protein family, as well as the Fos protein family or Jun protein family, and bind to AP1-binding sites TGAG/CTCA by their conserved basic region leucine zippers (bZIPs) motifs (Kim et al., 2021). Therefore, we generated a promoter combining CRE as well as AP1-bindin sites to increase the binding possibility of our ATF2-mRuby2 fusion protein. A further increase in promoter efficiency was achieved by not only including one but three of each binding motif, this enables signal amplification by increasing the possibility of interaction between ATF2 and our promoter. Finally, we constructed a miniCMV promoter, just containing the TATA-box and the Initiator-Sequence of the original CMV-promoter, downstream of our 3xCRE3xAP1-binding sites to ensure a functional and strong transcription when activated by ATF2, of the, in the composite part, downstream positioned reporter protein miRFP670.

Cloning

Theoretical Part Design

We generated a promoter sequence containing three CRE-binding motifs as well as three AP1-binding sites followed by the minimal CMV (miniCMV) promoter. The miniCMV promoter contains the TATA-box and the Initiator-Sequence of the original CMV-promoter, ensuring a successful transcription, but in parallel allowing for specific expression dependent on upstream laying individually chosen binding sites. The sequence was synthesized with approx. 20 bp-long overhangs to allow for correct orientation when integrated upstream of a reporter gene in a plasmid backbone.

Sequence and Features

Characterization

For further analysing co-expression experiments with PknB (K5317013) and the ATF2 (K5317017) please visit the registry entry (K5317022).

References

Hai, T. W., Liu, F., Coukos, W. J., & Green, M. R. (1989). Transcription factor ATF cDNA clones: An extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. Genes & Development, 3(12b), 2083–2090. https://doi.org/10.1101/gad.3.12b.2083

Kim, E., Ahuja, A., Kim, M. Y., & Cho, J. Y. (2021). DNA or Protein Methylation-Dependent Regulation of Activator Protein-1 Function. Cells, 10(2), 461. https://doi.org/10.3390/cells10020461

Kirsch, K., Zeke, A., Tőke, O., Sok, P., Sethi, A., Sebő, A., Kumar, G. S., Egri, P., Póti, Á. L., Gooley, P., Peti, W., Bento, I., Alexa, A., & Reményi, A. (2020). Co-regulation of the transcription controlling ATF2 phosphoswitch by JNK and p38. Nature Communications, 11(1), 5769. https://doi.org/10.1038/s41467-020-19582-3

Miller, M., Donat, S., Rakette, S., Stehle, T., Kouwen, T. R. H. M., 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