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The replication protein of pHW126 auto-controls its expression
1Biotechnology of Horticultural Crops, TUM School for Life Sciences Weihenstephan, Technische Universität München, Liesel-Beckmann-Straße 1, 85354 Freising-Weihenstephan, Germany.
Plasmid
|March 17, 2017
Summary
The replication gene (rep) expression in pHW126 rolling circle plasmids is auto-regulated. The Rep protein binds to direct repeats in the promoter, reducing its own transcription for an economic regulatory strategy.
Area of Science:
- Molecular Biology
- Plasmid Biology
- Gene Regulation
Background:
- pHW126 is a rolling circle plasmid where the replication gene (rep) is essential for autonomous replication.
- The regulatory mechanism controlling rep gene expression was previously unknown.
Purpose of the Study:
- To investigate the regulation of rep gene expression in pHW126.
- To determine if the Rep protein auto-regulates its own expression.
Main Methods:
- Sequence analysis of the rep promoter region.
- Reporter assays to measure promoter activity.
- Electrophoretic mobility shift assays (EMSA) to study protein-DNA interactions.
Main Results:
- A conserved region with three imperfect direct repeats (DR2.1, DR2.2, DR2.3) was identified in the rep promoter.
- These direct repeats function as transcriptional enhancers.
- The Rep protein binds to DR2.1 and DR2.3, reducing transcriptional activity.
- DR2.2 is not bound but influences the positioning of DR2.1 and DR2.3.
Conclusions:
- Rep gene expression in pHW126 is auto-regulated by the Rep protein.
- This auto-regulation involves the Rep protein binding to direct repeats in the promoter, acting as a negative feedback loop.
- This mechanism provides an economical strategy for regulating replication protein synthesis.
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