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pH-dependent autocleavage of lambda repressor occurs in the operator-bound form: characterization of lambda repressor
Kaushik Ghosh1, Atasi Pal, Rajagopal Chattopadhyaya
1Department of Biochemistry, Bose Institute, P-1/12, CIT Scheme VII M, Calcutta 700054, India.
The Biochemical Journal
|January 22, 2004
Summary
The spontaneous autocleavage of the lambda cI repressor was measured at various pH and temperatures. This process occurs in both operator-free and operator-bound states, suggesting proximity of catalytic and cleavage sites.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- The lambda cI repressor regulates the lysogenic state of the bacteriophage lambda.
- RecA-mediated cleavage is the primary pathway for repressor inactivation during the lytic cycle.
- Spontaneous autocleavage represents a RecA-independent pathway for repressor degradation.
Purpose of the Study:
- To quantify the rate constants of spontaneous, RecA-independent autocleavage of the lambda cI repressor.
- To investigate the influence of pH, temperature, and operator binding on autocleavage rates.
- To elucidate the structural and dynamic relationship between the catalytic and cleavage sites.
Main Methods:
- Measurement of first-order rate constants for lambda cI repressor autocleavage at pH 10.6, 9, and 8 across different temperatures (27, 37, 42 °C).
- Comparison of autocleavage rates in operator-free and operator-bound repressor states.
- Inhibition of autocleavage using phenylmethylsulfonyl fluoride (PMSF).
- Assessment of trans-cleavage activity using wild-type lambda repressor fragments.
Main Results:
- Spontaneous autocleavage rates were determined at pH 10.6, 9, and 8, decreasing with lower pH.
- Autocleavage occurred in both operator-free and operator-bound states, with rates comparable or higher in the bound state.
- PMSF treatment inhibited autocleavage, consistent with a catalytic serine or lysine residue.
- Type II trans-cleavage was not observed with wild-type repressor fragments, unlike mutant LexA repressors.
- The catalytic and cleavage sites are in close proximity in both operator-free and -bound wild-type lambda cI repressor.
Conclusions:
- The lambda cI repressor undergoes spontaneous autocleavage, a RecA-independent process.
- Operator binding does not significantly hinder, and can enhance, spontaneous autocleavage.
- The proximity of catalytic and cleavage sites is crucial for wild-type lambda cI repressor autocleavage.
- This slow, spontaneous cleavage may play a role in maintaining the lysogenic state in the absence of RecA activity.