SsrA-mediated peptide tagging caused by rare codons and tRNA scarcity

Area of Science:

• Molecular Biology
• Bacterial Genetics
• RNA Biology

Background:

• SsrA RNA (tmRNA) is a bacterial RNA molecule with dual tRNA and mRNA characteristics.
• It mediates the addition of a C-terminal peptide tag to proteins lacking in-frame stop codons.
• This tagging process typically targets aberrant proteins for degradation.

Purpose of the Study:

• To investigate the mechanism of SsrA RNA-mediated peptide tagging.
• To determine if ribosome stalling at rare codons is sufficient to activate SsrA tagging.
• To characterize the role of tRNA availability in SsrA tagging efficiency.

Main Methods:

• Design of a modified SsrA RNA variant to create a stable peptide tag.
• In vivo and in vitro assays to detect and analyze protein tagging.
• Manipulation of tRNA(AGA) levels to assess its impact on SsrA tagging.

Main Results:

• A stable peptide tag (AANDENYALDD) was successfully introduced onto model proteins lacking stop codons, detectable both in vivo and in vitro.
• Ribosome stalling at clusters of rare arginine codons (AGA) was demonstrated to be sufficient for recruiting and activating the SsrA peptide tagging system.
• Scarcity of the cognate tRNA(AGA) was identified as essential for efficient tagging at rare AGA codons; supplemental tRNA(AGA) suppressed tagging, while depletion enhanced it.
• Tagging at rare codons appears to occur at an internal mRNA site, not at the 3' end of cleaved mRNA.

Conclusions:

• The SsrA RNA system can tag proteins in response to ribosome stalling at rare codons, independent of stop codon absence.
• tRNA availability is a critical regulatory factor for SsrA-mediated peptide tagging at specific mRNA sequences.
• The findings provide insights into the intricate mechanisms of bacterial translational quality control and stress response.