The Rep protein binding elements of the plasmid ColE2-P9 replication origin.
The plasmid ColE2-P9 (ColE2) origin (32bp) is specifically recognized by the plasmid-specified Rep protein that initiates DNA replication. The ColE2 origin is divided into at least three functional subregions (I, II, and III), and three sites (a, b, and c) found in subregions I and II play important roles in Rep protein binding. We performed SELEX experiments of plasmid ColE2 to determine the optimal sequences for specific binding of the Rep protein. From these experiments, we obtained a common 16-bp sequence (5'-TGAGACCANATAAGCC-3'), which corresponds to about one half of the minimal ColE2 origin and contains sites a and b. Gel mobility shift assays using single-point mutant origins and the Rep protein further indicated that high affinity sequence-specific recognition by the Rep protein requires sites a, b, and c, but that mutations in site c were less disruptive to this recognition than those in sites a and b.
Yagura
Masaru
M
Department of Biology, Faculty of Science, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan.
Itoh
Tateo
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D004261:DNA Replication / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D018741:Replication Origin / Q000235:genetics*; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D029968:Escherichia coli Proteins; D005656:Fungal Proteins; C051237:REP protein, yeast plasmid; C512429:Rep 35-kDa protein, E coli; D015534:Trans-Activators