title: pH-dependent autocleavage of lambda repressor occurs in the operator-bound form: characterization of lambda repressor autocleavage.
abstract: 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.
authors.affiliation: Department of Biochemistry, Bose Institute, P-1/12, CIT Scheme VII M, Calcutta 700054, India.
authors.forename: Kaushik
authors.initials: K
authors.lastname: Ghosh
authors.forename: Atasi
authors.initials: A
authors.lastname: Pal
authors.forename: Rajagopal
authors.initials: R
authors.lastname: Chattopadhyaya
mesh_terms: D020134:Catalytic Domain; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009875:Operator Regions, Genetic; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature; D014105:Tosyl Compounds / Q000494:pharmacology; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014105:Tosyl Compounds; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; C003614:4-toluenesulfonyl fluoride