The structure and functions of protein RepA of plasmid Rtsl

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RepA protein, encoded on the mini-Rtsl geneome and essential for the plasmid Rtsl replication, is composed of 288 amino acid residues.1. Purification of RepA and binding of the protein to DNA.Purified RepA was prepared from the E. coli cell-lysate harboring the recombinant plasmid with repA. Binding of the purified RepA to mini-Rtsl DNA was studied by DNaseI foot printing method, and revealed that RepA binds specifically to inci and incII iterons as well as to the immediately upstream of the repa promoter.2. Construction of repa mutants and their altered functions.1) Mutations in the middle of repA. We utilized the unique StyI and XbaI sites that located in the middle of repA gene to insert a 4-amino-acid in frame to RepA protein. Both RepA mutant proteins lost initiator function but retained strong autorepressor-incompatibility functions.2) Mutations in the 3' end of repA. Seven RepA mutants, each of which contained a single amino acid substitution or small deletion in the C-terminal region of RepA, were obtained by site-directed mutagenesis, The following findings were obtained.(a) Lys268 is important for both initiator and autorepressor-incompatibility functions. (b) Arg279 is involved in only initiator function. (c) Deletion of four amino acid residues from the C-terminus induced a high copy number of the plasmid. (d) Deletion of five amino acid residues from the C-terminus restored the wild type RepA phenotypes.