Replication of an RK2 miniplasmid derivative in vitro by a DNA/membrane complex extracted from Escherichia coli: involvement of the dnaA but not dnaK host proteins and association of these and plasmid-encoded proteins with the inner membrane.
A DNA/membrane complex extracted from a miniplasmid derivative of the broad host range plasmid RK2 cultured in Escherichia coli capable of synthesizing new plasmid supercoiled DNA in vitro was treated with antibodies that were made against or reacted with the dnaA and dnaK host-encoded proteins, respectively. Anti-dnaA protein antibody inhibited total plasmid DNA synthesis significantly and the synthesis of supercoil plasmid DNA almost completely. In contrast, anti-dnaK protein antibody and nonimmune serum had little or no effect on total plasmid DNA synthesis. Both proteins were found to be present in the inner but not outer membrane fraction of E. coli. A variety of miniplasmid-encoded proteins which had previously been found in the DNA/membrane complex have also been localized to the inner but not outer membrane fraction. These include an essential initiation protein of 32 kDa (and an overlapping protein of 43 kDa coded for by the same gene), as well as a 30-kDa protein that may be linked to incompatibility functions. Various extraction methods were used to distinguish between the associated and the integral nature of the plasmid-encoded proteins. The results demonstrated that the essential replication proteins (32 and 43 kDa) as well as the 30-kDa protein was tightly bound to the inner membrane. Computer analysis of the amino acid sequence of the 32 (and 43)-kDa protein revealed a hydrophobic region that is only half that normally required to span the membrane. Other interactions are discussed with respect to attaching this protein to the membrane.
Kostyal
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DA
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06457.
Farrell
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M
McCabe
A
A
Mei
Z
Z
Firshein
W
W
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics*; D002462:Cell Membrane / Q000032:analysis; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics; D015151:Immunoblotting; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010957:Plasmids; D001616:beta-Galactosidase / Q000378:metabolism
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D009247:NADH, NADPH Oxidoreductases; D001616:beta-Galactosidase; C063842:dnaK protein, E coli