Revision as of 18:04, 1 October 2024

3xCre3xAP1-miniCMV Promoter

Usage and Biology

In order 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 for example 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 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 motifs, this enables a 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-bining 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