Isolation and characterization of DNA-binding mutants of a plasmid replication initiation protein utilizing an in vivo binding assay.

An in vivo screen was developed for the identification of mutants of the RK2 replication initiation protein, TrfA, that were altered in their binding to the iterons within the plasmid RK2 origin of replication. This assay is based on an antibiotic selection system originally described by Elledge, Sugiono, Guarente, and Davis (Proc. Natl. Acad. Sci. USA86, 3689-3693, 1989) for the isolation in vivo of genes encoding sequence-specific DNA-binding proteins. A TrfA-specific binding site consisting of two 17-bp iterons separated by a nonrandom 6-bp spacer was placed 3' to a strong constitutive promoter. This promoter-iteron fragment was then inserted into the assay vector convergent to the aadA gene such that an increased level of spectinomycin resistance by the Escherichia coli host was dependent on the binding of wild-type TrfA protein to the binding site. The in vivo system was used to specifically isolate TrfA mutants which were either defective in binding or capable of effecting increased levels of spectinomycin resistance as compared to wild-type TrfA. The defective TrfA mutants isolated by this screen were purified and found to be considerably less effective in DNA binding by in vitro gel mobility shift assays. The map location was determined for these six defective TrfA mutants. Each of the mutations consisted of a single base change and mapped within codons extending over a 162 amino acid sequence. All of the mutants which were capable of effecting increased levels of spectinomycin resistance in the in vivo DNA-binding assay also showed some alteration in RK2 replication in vivo with most of the mutants having a copy-up phenotype similar to previously isolated TrfA mutants able to maintain an eight-iteron RK2 origin plasmid at a higher copy number.