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The mechanism of the stringent control of lambda plasmid DNA replication
A Szalewska-Pałasz1, A Wegrzyn, A Herman
1Department of Molecular Biology, University of Gdańsk.
The EMBO Journal
|December 1, 1994
Summary
Amino acid starvation inhibits lambda plasmid DNA replication in wild-type Escherichia coli via the stringent response. This occurs because ppGpp interferes with transcriptional activation of the ori lambda site, halting replication.
Area of Science:
- Molecular Biology
- Bacteriology
- Genetics
Background:
- Lambda plasmid DNA replication in Escherichia coli is regulated by cellular conditions.
- The stringent response, triggered by amino acid starvation in wild-type E. coli, affects various cellular processes, including DNA replication.
- The relaxed response in relA mutants exhibits different regulatory mechanisms compared to the stringent response.
Purpose of the Study:
- To investigate the mechanism of lambda plasmid DNA replication inhibition during amino acid starvation in wild-type E. coli.
- To determine the role of the stringent and relaxed responses in regulating lambda plasmid replication.
- To elucidate the involvement of ppGpp and transcriptional activation in this regulatory process.
Main Methods:
- Comparative analysis of lambda plasmid replication in stringent (wild-type) and relaxed (relA mutant) E. coli strains under amino acid starvation.
- Investigation of transcription from the pR promoter, a key site for replication initiation, using rifampicin treatment.
- Genetic manipulation by replacing the pR promoter with plac to assess the effect of ppGpp insensitivity on replication.
Main Results:
- Lambda plasmid replication was inhibited in amino acid-starved wild-type E. coli (stringent response) but not in relA mutants (relaxed response).
- Transcription from pR, essential for replication protein synthesis and ori lambda activation, was significantly decreased during the stringent response and with increased ppGpp levels.
- Replacing pR with plac restored lambda plasmid DNA replication under both stringent and relaxed conditions, as well as during ppGpp overproduction.
Conclusions:
- The inhibition of lambda plasmid DNA replication in amino acid-starved wild-type E. coli is mediated by ppGpp.
- ppGpp exerts its inhibitory effect by suppressing the transcriptional activation of the ori lambda site.
- The pR promoter's sensitivity to ppGpp is a critical factor in the stringent response-induced replication block.