kfrA gene of broad host range plasmid RK2 encodes a novel DNA-binding protein.
The korABF operon of broad host range IncP plasmid RK2 encodes proteins that coordinate expression of many other operons and that aid plasmid stability by providing at least part of a partitioning apparatus. The kfrA gene lies downstream from this operon and its transcription is repressed by all except one of the proteins encoded by this operon (KorA, KorFI, KorFII and KorB). We report here that transcription from the kfrA promoter is autoregulated by the kfrA gene product. We have purified KfrA, which is an acidic polypeptide of 308 amino acid residues, and show that it is a site-specific DNA-binding protein whose operator overlaps the primary kfrA promoter. Deletion analysis suggests that this activity is critically dependent on the N-terminal section of KfrA, which appears to contain an alpha-helix-beta-turn-alpha-helix motif. Circular dichroism confirmed the structural prediction that KfrA is almost entirely alpha-helical. The position of predicted turns suggests that, while amino acid residues 1 to 80 may form a globular domain of four or five helices, residues 80 to 280 of KfrA may adopt an extended coiled-coil domain containing a heptad repeat segment, which is probably responsible for formation of the multimers detected by crosslinking. The possibility that this unusual structure serves a second function, for example in providing a bridge to host structures required for plasmid partitioning, is discussed.
Jagura-Burdzy
G
G
School of Biological Sciences, University of Birmingham, U.K.
Thomas
C M
CM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009875:Operator Regions, Genetic; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D013329:Structure-Activity Relationship
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger