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Structure of a hyper-cleavable monomeric fragment of phage lambda repressor containing the cleavage site region.

ABSTRACT: 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.

SUBMITTER: Ndjonka D

PROVIDER: S-EPMC1896146 | biostudies-literature | 2006 Sep

REPOSITORIES: biostudies-literature

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Structure of a hyper-cleavable monomeric fragment of phage lambda repressor containing the cleavage site region.

Ndjonka Dieudonné D Bell Charles E CE

Journal of molecular biology 20060715 3

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 monome ...[more]

PMID: 16934834

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Project description:The first-order rate constants for the RecA-independent, spontaneous, pH-dependent autocleavage of the lambda cI repressor was measured in the present study at pH 10.6 at 27, 37 and 42 degrees C respectively. Autocleavage of the repressor occurs also at pH 9 and 8, although at progressively slower rates. We demonstrate that the spontaneous autocleavage occurs also in the operator-bound state, at a rate either higher than or equal to the rate in solution, depending on the pH value. Owing to the near equality of the rate constant in both operator-free and operator-bound repressors, it can be inferred that the cleavage site has a similar structure and dynamics with respect to the catalytic site in both forms at neutral pH. Covalent modification using PMSF, brought about by a large molar excess of the reagent, inhibits autocleavage of the lambda repressor. The difficulty in obtaining this covalent modification is rationalized using our recent lambda repressor models. Bimolecular type II trans -cleavage was observed previously for mutant LexA repressors lacking a crucial catalytic serine or lysine residue [Kim and Little (1993) Cell (Cambridge, Mass.) 73, 1165-1173], but it could still be cleaved by an 85-202 'enzyme' fragment possessing an improved or hypercleavable character lacking its own cleavage site. Such a type II trans -cleavage was not observed with the covalently modified intact lambda repressor used as substrate and the purified wild-type lambda repressor 112-236 fragment used as the 'enzyme'. All these results show that for the wild-type lambda repressor, the catalytic site is close to the cleavage site in both operator-free and -bound states. In the lytic pathway, the repressor is mainly cleaved via RecA-mediated cleavage, which occurs much faster than the spontaneous autocleavage; the possible biological significance of this slow, spontaneous, but constant, autocleavage is related to the lysogenic state, when RecA-mediated cleavage is absent.

Dataset Information

Structure of a hyper-cleavable monomeric fragment of phage lambda repressor containing the cleavage site region.

Publications

Structure of a hyper-cleavable monomeric fragment of phage lambda repressor containing the cleavage site region.

Similar Datasets

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