IS911 transposition is regulated by protein-protein interactions via a leucine zipper motif.
Efficient intermolecular transposition of bacterial insertion sequence IS911 involves the activities of two element-encoded proteins: the transposase, OrfAB, and a regulatory factor, OrfA. OrfA shares the majority of its amino acid sequence with the N-terminal part of OrfAB. This includes a putative helix-turn-helix and three of four heptads of a leucine zipper motif. OrfA strongly stimulates OrfAB-mediated intermolecular transposition both in vivo and in vitro. The present results support the notion that this is accomplished by direct interaction between these two proteins via the leucine zipper. We used both a genetic approach, based on gene fusions with phage lambda repressor, and a physical approach, involving co-immunoprecipitation, to show that OrfA not only undergoes oligomerisation but is capable of engaging with OrfAB to form heteromultimers, and that the leucine zipper is necessary for both types of interaction. Furthermore, mutation of the leucine zipper in OrfA inactivated its regulatory function. Previous observations demonstrated that the integrity of the leucine zipper motif was also important for OrfAB binding to the IS911 terminal inverted repeats. Here, we show, in gel shift experiments, using a derivative of OrfAB deleted for the C-terminal catalytic domain, OrfAB[1-149], that the protein is capable of pairing two inverted repeats to generate a species resembling a "synaptic complex". Preincubation of OrfAB[1-149] with OrfA dramatically reduced formation of this complex and favored formation of an alternative complex devoid of OrfA. Together these results suggest that OrfA exerts its regulatory effect by interacting transiently with OrfAB via the leucine zipper and modifying OrfAB binding to the inverted repeats.
Haren
L
L
Laboratoire de Microbiologie et Génétique Moléculaire, CNRS Université Paul Sabatier, 118 Route de Narbonne, Toulouse, 31062, France.
Normand
C
C
Polard
P
P
Alazard
R
R
Chandler
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology; D017124:Conserved Sequence / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005807:Genes, Overlapping / Q000235:genetics; D016350:Leucine Zippers / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D011233:Precipitin Tests; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D019895:Transposases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D019895:Transposases