title: The replication protein of pHW126 auto-controls its expression.
abstract: pHW126 belongs to a small group of rolling circle plasmids. So far, the region mediating autonomous replication has been identified and it was shown that the rep gene is required for replication. However, the regulation of rep expression remained elusive. Here evidence is presented that expression of the replication gene rep is auto-regulated. Sequence analysis revealed a conserved stretch in the rep promoter consisting of three imperfect direct repeats (DR2.1, DR2.2 and DR2.3). Assays for promoter activity showed that these direct repeats act as an enhancer of transcriptional activity. Interestingly, the activating effect was reduced in the presence of Rep protein. Electrophoretic mobility shift assays demonstrated that the Rep protein can directly bind to direct repeats DR2.1 and DR2.3 while DR2.2 is not bound but places DR2.1 and DR2.3 in an appropriate distance. These results show that the synthesis of Rep protein is auto-regulated. In the absence of Rep protein the promoter is, due to the presence of the direct repeats acting as a transcriptional enhancer, highly active. Binding of Rep to the direct repeats reduces the transcription rate significantly. Since this regulation mechanism is independent of a specialised regulator protein it is presumably a very economic strategy.
authors.affiliation: Biotechnology of Horticultural Crops, TUM School for Life Sciences Weihenstephan, Technische Universität München, Liesel-Beckmann-Straße 1, 85354 Freising-Weihenstephan, Germany. Electronic address: wilfried.rozhon@wzw.tum.de.
authors.forename: Wilfried
authors.initials: W
authors.lastname: Rozhon
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020638:Rahnella / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases
keywords: DNA binding protein; Gene expression; Plasmid; Rolling circle replication; Transcriptional enhancer; pHW126