Analysis of the CoIE1 stability determinant Rcd

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• Multicopy plasmid instability is often caused by multimer formation.
• Plasmid CoIE1 utilizes Xer-mediated recombination at the cer site for stable monomerization.
• However, plasmid stability also depends on a promoter (Pcer) within the cer site, independent of recombination.

Purpose of the Study:

• To investigate the role of the Pcer promoter and its transcript (Rcd) in plasmid CoIE1 stability.
• To characterize the Rcd transcript, including its transcription initiation, degradation, and secondary structure.
• To explore the mechanism of Rcd's function, including its potential as an antisense RNA or protein-interacting molecule.

Main Methods:

• Transcriptional mapping to determine the 5' and 3' termini of the Rcd transcript.
• RNA folding analysis and secondary structure prediction for wild-type and mutant Rcd transcripts.
• Site-directed mutagenesis to identify critical regions of the rcd gene and Pcer promoter.
• Bioinformatic analysis (DNA hybridization and sequence analysis) to search for Rcd targets in the E. coli chromosome.

Main Results:

• Transcription initiation from Pcer was confirmed, with the Rcd transcript showing heterogeneity at its 3' terminus.
• Mutations affecting Rcd's growth inhibition activity were mapped to Pcer or the rcd coding sequence, altering transcript levels or secondary structure.
• Analysis of cer-like sites revealed conserved Pcer-like sequences and similar predicted Rcd structures.
• No plausible antisense targets for Rcd were found in the E. coli chromosome.

Conclusions:

• Plasmid CoIE1 stability is regulated by both Xer-mediated recombination and the Pcer-Rcd system.
• The Rcd transcript's secondary structure is critical for its function in inhibiting cell division.
• Rcd likely functions by interacting with a protein target rather than acting as an antisense RNA.