Intramolecular Interactions Dominate the Autoregulation of Escherichia coli Stringent Factor RelA

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Amino acid starvation in Escherichia coli activates the stringent factor RelA, producing the alarmone nucleotide (p)ppGpp.
• The alarmone (p)ppGpp is crucial for bacterial stress adaptation, regulating transcription, translation, and metabolism.
• RelA's enzymatic activity is allosterically controlled by ligands and its own domains, with proposed auto-regulation mechanisms including cis and trans inhibition.

Purpose of the Study:

• To investigate the regulatory roles of individual domains within E. coli RelA.
• To determine if RelA dimerization is the key mechanism for its auto-regulation.
• To elucidate the function of the RelA C-terminal domain (CTD) and its RRM domain.

Main Methods:

• Ectopic expression of RelA CTD in E. coli.
• Biochemical assays to assess RelA enzymatic activity at varying concentrations.
• Analysis of RelA mutants, including deletion of the RRM domain in the CTD.

Main Results:

• Ectopic expression of RelA CTD did not inhibit native RelA activation, suggesting auto-regulation is not via inhibitory complex formation.
• Enzyme activity did not decrease with increasing RelA concentration, contradicting efficient auto-inhibition through dimerization.
• High-level CTD expression inhibited growth by directly suppressing protein synthesis, independent of native RelA.
• Deletion of the CTD's RRM domain caused growth inhibition due to (p)ppGpp accumulation, indicating de-regulated synthetic activity.

Conclusions:

• RelA dimerization is not the primary mechanism for auto-regulation in E. coli.
• The RelA CTD directly inhibits protein synthesis, likely by interacting with the ribosomal A-site.
• The RRM domain within the CTD is essential for controlling RelA's synthetic activity and preventing aberrant (p)ppGpp accumulation.