A base-paired hairpin structure essential for the functional priming signal for DNA replication of the broad host range plasmid RSF1010

The two single-strand DNA initiation signals, ssiA(RSF1010) and ssiB(RSF1010) of the broad host-range plasmid RSF1010 contain proposed stem-loop structures. Nine single base-change mutations in the stem of the ssiA structure, each of which destroyed a relevant base pairing, damaged the ssiA activity. A second single-base change was introduced into each of the nine ssiA mutants in such a way that the base pairing was restored. Only three out of nine second base changes that restored the base pairing restored the ssiA activity up to the wild-type level. Thus, the three are intramolecular suppressors. The results strongly suggested that, in the area of the stem of ssiA where the suppressor mutations fell, base pairing was the most important structural parameter for the ssiA activity. By contrast, it is most probable that, in the other part of the stem of ssiA, both base-pairing and the intrinsic base sequence were the major determinants of the ssiA activity.