Nucleic Acids Research, 1992, Vol. 20, No. 8 1851-1858
© 1992
MOLECULAR BIOLOGY |
KorB protein of promiscuous plasmid RP4 recognizes inverted sequence repetitions in regions essential for conjugative plasmid transfer
Max-Planck-lnstitut für Molekulare Genetik Ihnestrasse 73, D-1000 Berlin 33, Germany
*To whom correspondence should be addressed
Received February 21, 1992. Revised March 20, 1992. Accepted March 20, 1992.
We have constructed a RP4 KorB overproducing strain and purified the protein to near homogeneity. KorB is a DNA binding protein recognizing defined paiindromic 13-bp sequences (TTTAGCSGCTAAA). Inverted sequence repetitions of this type, designated OB, are present on RP4 12 times. OB-sequences are localized In replication and maintenance regions as well as in the regions Tra1 and Tra2 essential for conjugative transfer. All sites found In Tra regions by computer search act as targets for specific binding of KorB protein. KorB-DNA complexes were detected by DNA fragment retardation assay using polyacrylamide gels. The 13-bp symmetric arrangement of the consensus Oe-sequence constitutes the core for binding KorB protein since any truncation of this sequence prevents complex assembly or leads to a considerable destabilizatlon of the KorB-DNA complexes. A hydroxyl radical footprint analysis demonstrated complex formation of KorB with the Oe-sequence directly and suggests the presence of an unusual DNA structure within the nucleoprotein complex.
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