A leucine zipper motif determines different functions in a DNA replication protein.
RepA is the replication initiator protein of the Pseudomonas plasmid pPS10 and is also able to autoregulate its own synthesis. Here we report a genetic and functional analysis of a leucine zipper-like (LZ) motif located at the N-terminus of RepA. It is shown that the LZ motif modulates the equilibrium between monomeric and dimeric forms of the protein and that monomers of RepA interact with sequences at the origin of replication, oriV, while dimers are required for interactions of RepA at the repA promoter. Further, different residues of the LZ motif are seen to have different functional roles. Leucines at the d positions of the putative alpha-helix are relevant in the formation of RepA dimers required for transcriptional autoregulation. They also modulate other RepA-RepA interactions that result in cooperative binding of protein monomers to the origin of replication. The residues at the b/f positions of the putative helix play no relevant role in RepA-RepA interactions. These residues do not affect RepA autoregulation but do influence replication, as demonstrated by mutants that, without affecting binding to oriV, either increase the host range of the plasmid or are inactive in replication. It is proposed that residues in b/f positions play a relevant role in interactions between RepA and host replication factors.
Garcia de Viedma
D
D
Departamento de Microbiologia Molecular, Centro de Investigaciones Biologicas, Madrid, Spain.
Giraldo
R
R
Rivas
G
G
Fernández-Tresguerres
E
E
Diaz-Orejas
R
R
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000502:physiology*; D001483:Base Sequence; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000502:physiology; D015964:Gene Expression Regulation, Bacterial; D016350:Leucine Zippers; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009875:Operator Regions, Genetic; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; C074165:lemA protein, bacterial