Functional characterization of a replication initiator protein.
Functional domains in the RepI replication initiator protein have been identified by classical and site-directed mutagenesis techniques. Mutations conferring an increase in plasmid copy number contained alterations in a key position of a putative helix-turn-helix DNA binding motif. The mutations did not appear to affect autorepressing functions. Regions of RepI important for autorepression were localized as well. Two classes of mutations resulting in diminished autorepression functions were identified. One class was distinguished by an elevated copy number, while the other class remained at the wild-type copy number level. Analysis of the various mutations leading to changes in copy number or autorepressing functions suggest that in some cases the autorepression and initiating functions of the RepI protein are separable. Finally, analysis with deletion clones suggests that the trans-acting autorepressing functions of RepI might depend on intermolecular coupling control.
Department of Microbiology and Immunology, Oregon Health Sciences University, Portland 97201.
A E
AE
Gammie
M E
ME
Tolmasky
J H
JH
Crosa
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D003086:Bacteriocin Plasmids / Q000235:genetics*; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000235:genetics; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D015534:Trans-Activators
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C069089:RepI protein, Plasmid ColV-K30; D012097:Repressor Proteins; D015534:Trans-Activators