Structure of a hyper-cleavable monomeric fragment of phage lambda repressor containing the cleavage site region.
The key event in the switch from lysogenic to lytic growth of phage lambda is the self-cleavage of lambda repressor, which is induced by the formation of a RecA-ssDNA-ATP filament at a site of DNA damage. Lambda repressor cleaves itself at the peptide bond between Ala111 and Gly112, but only when bound as a monomer to the RecA-ssDNA-ATP filament. Here we have designed a hyper-cleavable fragment of lambda repressor containing the hinge and C-terminal domain (residues 101-229), in which the monomer-monomer interface is disrupted by two point mutations and a deletion of seven residues at the C terminus. This fragment crystallizes as a monomer and its structure has been determined to 1.8 A resolution. The hinge region, which bears the cleavage site, is folded over the active site of the C-terminal oligomerization domain (CTD) but with the cleavage site flipped out and exposed to solvent. Thus, the structure represents a non-cleavable conformation of the repressor, but one that is poised for cleavage after modest rearrangements that are presumably stabilized by binding to RecA. The structure provides a unique snapshot of lambda repressor in a conformation that sheds light on how its self-cleavage is tempered in the absence of RecA, as well as a framework for interpreting previous genetic and biochemical data concerning the RecA-mediated cleavage reaction.
Ndjonka
Dieudonné
D
Department of Molecular and Cellular Biochemistry, Ohio State University College of Medicine, Columbus, OH 43210, USA.
Bell
Charles E
CE
D010582:Bacteriophage lambda / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008967:Molecular Biology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D017510:Protein Folding; D011938:Rec A Recombinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D055672:Static Electricity; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D011938:Rec A Recombinases