Chapter title |
DNA-protein interaction at the replication origins of plasmid chromosomes.
|
---|---|
Chapter number | 29 |
Book title |
Plasmids in Bacteria
|
Published in |
Basic life sciences, January 1985
|
DOI | 10.1007/978-1-4613-2447-8_29 |
Pubmed ID | |
Book ISBNs |
978-1-4612-9487-0, 978-1-4613-2447-8
|
Authors |
D. Bastia, C. Vocke, J. Germino, J. Gray, Bastia, D., Vocke, C., Germino, J., Gray, J. |
Abstract |
Novel techniques have been developed to purify replication initiator proteins of the plasmids R6K and pSC101. The techniques consist of tagging the initiator cistrons at the C-terminus with beta-galactosidase-encoding DNA of Escherichia coli in the correct translational phase. The hybrid proteins are then rapidly purified by adsorption to and elution from a beta-galactosidase- specific affinity column. Two procedures have been devised to isolate the nonfused initiator proteins using the fused protein as a handle. The first procedure, called subunit association chromatography, exploits the association of a monomer of nontagged protein with that of beta-galactosidase-tagged protein in isolating both types of proteins by beta-galactosidase specific affinity column chromatography. The second procedure involves the fusion of the initiator protein to beta-galactosidase via a specific linker DNA. The linker DNA encodes a protein which is readily and specifically hydrolyzed by a sequence specific protease, thus releasing the initiator protein from beta-galactosidase. Using purified or partially purified initiator protein, we have demonstrated that the R6K encoded initiator protein (Pi protein) binds to a consensus 22 bp sequence at 2 regions of the plasmid chromosome. The pSC101-encoded initiator protein binds to sequences at or near the plasmid replication origin. At low concentrations the protein binds to a nucleation site and upon raising the concentrations of the protein binding is promoted at 4 adjacent sequences that have partial homologies with the nucleation sequence. Deletion of the binding site leads to a nonfunctional replication origin. |
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