Altered (copy-up) forms of initiator protein pi suppress the point mutations inactivating the gamma origin of plasmid R6K.
The R6K gamma origin core contains the P2 promoter, whose -10 and -35 hexamers overlap two of the seven binding sites for the R6K-encoded pi protein. Two mutations, P2-201 and P2-203, which lie within the -35 region of P2, are shown to confer a promoter-down phenotype. We demonstrate here that these mutations prevent replication of a gamma origin core plasmid. To determine whether or not the reduced promoter activity caused by these mutations is responsible for their effect on replication, we generated two new mutations (P2-245-6-7 and P2-246) in the -10 hexamer of the P2 promoter. Although these new mutations inhibit P2 activity as much as the P2-201 and P2-203 mutations, they do not prevent replication of the gamma origin core. Therefore, activity of the P2 promoter does not appear to be required for replication. We also show that the inability of the gamma origin to function in the presence of the P2-201 and P2-203 mutations is reversed by the hyperactive variants of pi protein called copy-up pi. This suppression occurs despite the fact that in vivo dimethyl sulfate methylation protection patterns of the gamma origin iterons are identical in cells producing wild-type pi and those producing copy-up pi variants. We discuss how the P2-201 and P2-203 mutations could inhibit replication of the gamma origin core and what mechanisms might allow the copy-up pi mutants to suppress this deficiency.
Urh
M
M
Department of Bacteriology, University of Wisconsin, Madison 53706, USA.
Flashner
Y
Y
Shafferman
A
A
Filutowicz
M
M
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004265:DNA Helicases; D004261:DNA Replication; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics*; D014644:Genetic Variation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010448:Peptide Initiation Factors / Q000235:genetics*; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011815:R Factors / Q000096:biosynthesis / Q000235:genetics*; D018741:Replication Origin; D017422:Sequence Analysis, DNA; D013489:Suppression, Genetic; D015534:Trans-Activators / Q000235:genetics*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D010448:Peptide Initiation Factors; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases