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Nucleic Acids Research, 1993, Vol. 21, No. 21 4900-4903
© 1993

MOLECULAR BIOLOGY

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

Dang-Ming Miao, Yoichi Honda, Katsunori Tanaka, Akihisa Higashi, Toshiaki Nakamura, Yoshitomo Taguchi, Hiroshi Sakai, Tohru Komano* and Michael Bagdasarian1

Laboratory of Biochemistry, Department of Agricultural Chemistry, Kyoto University Kyoto 606, Japan 1Department of Microbiology, Michigan State University East Lansing, Ml 48824, USA

*To whom correspondence should be addressed

Received August 3, 1993. Revised September 13, 1993. Accepted September 13, 1993.

The two single-strand DNA initiation signals, ssiA(RSF1010) and ssiB(RSF1010) of the broad hostrange 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, inthe 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.


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