Regulation of a muralytic enzyme-encoding gene by two non-coding RNAs

Area of Science:

• Microbiology
• Molecular Biology
• Gene Regulation

Background:

• Non-coding RNAs regulate gene expression post-transcriptionally.
• The rpfA gene, crucial for dormancy in streptomycetes, is associated with regulatory RNA elements.
• The mechanism of the rpfA riboswitch and its regulation by cyclic di-AMP were unknown.

Purpose of the Study:

• To elucidate the mechanism of the rpfA riboswitch in Streptomyces coelicolor.
• To investigate the role of the antisense small RNA (sRNA) Scr3097 in rpfA gene regulation.
• To understand the interplay of multiple regulatory RNAs controlling rpfA expression.

Main Methods:

• In vitro transcription assays to study riboswitch function.
• Extensive mutational analysis of the riboswitch and sRNA.
• Northern blotting to detect and quantify RNA levels.
• Growth in liquid and plate cultures to assess regulatory roles.

Main Results:

• The rpfA riboswitch mediates premature transcription termination in response to cyclic di-AMP.
• Ligand binding and an extended stem-loop are essential for riboswitch-mediated attenuation.
• The antisense sRNA Scr3097 accumulates with rpfA mRNA and is detected in specific growth phases.
• Scr3097 was found to increase rpfA mRNA abundance.

Conclusions:

• Multiple non-coding RNAs, including a riboswitch and an antisense sRNA, cooperatively regulate rpfA gene expression in streptomycetes.
• The rpfA riboswitch utilizes an unusual mechanism for cyclic di-AMP-dependent transcriptional control.
• The antisense sRNA Scr3097 plays a positive regulatory role in rpfA expression during specific developmental stages.