CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage

Area of Science:

• Bacteriology
• Molecular Biology
• Gene Regulation

Background:

• The Csr (carbon storage regulator) system is a global regulator influencing hundreds of Escherichia coli genes.
• CsrA protein typically represses translation by binding mRNA, but its activation mechanisms are less understood.
• CsrA is known to activate flhDC expression, which is crucial for flagellar biosynthesis and chemotaxis.

Purpose of the Study:

• To elucidate the mechanism by which CsrA activates flhDC gene expression.
• To identify and characterize CsrA binding sites on the flhDC mRNA.
• To investigate the role of RNase E in flhDC mRNA regulation by CsrA.

Main Methods:

• Computer modeling, gel mobility shift assays, and footprinting to identify CsrA binding sites.
• Analysis of flhD'-'lacZ expression under different mutation conditions.
• In vitro assays using RNase E to assess CsrA's protective effect on flhDC mRNA.
• Primer extension studies to identify mRNA decay intermediates and RNase E cleavage sites in vivo.

Main Results:

• Two CsrA binding sites (BS1 and BS2) were identified on the flhDC leader transcript.
• Mutations in csrA or binding sites reduced flhD'-'lacZ expression.
• CsrA protected flhDC mRNA from RNase E cleavage in vitro, dependent on BS1 and BS2.
• Deletion of RNase E cleavage sites increased flhD'-'lacZ expression, indicating a role for RNase E in regulation.

Conclusions:

• CsrA activates flhDC expression by inhibiting 5' end-dependent RNase E cleavage of the mRNA.
• This study reveals a novel mechanism of gene activation mediated by mRNA protection from degradation.
• Understanding CsrA's regulatory role is key to comprehending flagellar gene expression in Escherichia coli.