PreprintArticleVersion 1Preserved in Portico This version is not peer-reviewed
Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions
Version 1
: Received: 5 April 2024 / Approved: 6 April 2024 / Online: 8 April 2024 (05:43:18 CEST)
How to cite:
Salinas, P.; Bibak, S.; Cantos, R.; Tremiño, L.; Jerez, C.; Mata, T.; Contreras, A. Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions. Preprints2024, 2024040461. https://doi.org/10.20944/preprints202404.0461.v1
Salinas, P.; Bibak, S.; Cantos, R.; Tremiño, L.; Jerez, C.; Mata, T.; Contreras, A. Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions. Preprints 2024, 2024040461. https://doi.org/10.20944/preprints202404.0461.v1
Salinas, P.; Bibak, S.; Cantos, R.; Tremiño, L.; Jerez, C.; Mata, T.; Contreras, A. Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions. Preprints2024, 2024040461. https://doi.org/10.20944/preprints202404.0461.v1
APA Style
Salinas, P., Bibak, S., Cantos, R., Tremiño, L., Jerez, C., Mata, T., & Contreras, A. (2024). Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions. Preprints. https://doi.org/10.20944/preprints202404.0461.v1
Chicago/Turabian Style
Salinas, P., Trinidad Mata and Asunción Contreras. 2024 "Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions" Preprints. https://doi.org/10.20944/preprints202404.0461.v1
Abstract
Yeast two-hybrid approaches, which are based on fusion proteins that must co-localise to the nucleus to reconstitute the transcriptional activity of GAL4 have greatly contributed to understand the nitrogen interaction network of cyanobacteria, whose main hubs are the trimeric PII and the monomeric PipX regulators. The bacterial two-hybrid system, based on the reconstitution in the E. coli cytoplasm of the adenylate cyclase of Bordetella pertussis, should provide a relatively faster and presumably more physiological assay for cyanobacterial proteins than the yeast system. Here we use the bacterial two-hybrid system to gain additional insights into the cyanobacterial PipX interaction network while simultaneously assessing the advantages and limitations of the two most popular two-hybrid systems. Comprehensive mutational analysis of PipX and bacterial two-hybrid assays were performed to compare outcomes between yeast and bacterial systems. We detected interactions previously recorded in the yeast two-hybrid system as negative as well as a “false positive”, the self-interaction of PipX, which is rather an indirect interaction dependent on PII homologs from the E. coli host, a result confirmed by Western analysis with relevant PipX variants. This is, to our knowledge, the first report of the molecular basis of a false positive in the bacterial two-hybrid system.
Biology and Life Sciences, Biochemistry and Molecular Biology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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