Trans-translation mediated by Bacillus subtilis tmRNA

Area of Science:

• Molecular Biology
• Bacterial Genetics

Background:

• Trans-translation is a cellular mechanism that rescues ribosomes stalled on mRNA lacking stop codons.
• This process involves a transfer messenger RNA (tmRNA) and a protein tag, typically facilitated by ribosomal protein S1.
• Ribosomal protein S1 is absent in some Gram-positive bacteria, raising questions about trans-translation efficiency in these organisms.

Purpose of the Study:

• To investigate the functionality of trans-translation using tmRNA from Bacillus subtilis, a Gram-positive bacterium lacking ribosomal protein S1.
• To determine if the tmRNA from B. subtilis can produce tagged polypeptides in both its native host and in Escherichia coli.

Main Methods:

• Expression of a truncated gene lacking a termination codon in Bacillus subtilis.
• Co-expression of the truncated gene with Bacillus subtilis tmRNA in Escherichia coli.
• Analysis of C-terminal tag-peptide sequences in the resulting polypeptides.
• In vitro assessment of Bacillus subtilis tmRNA activity in an E. coli system.

Main Results:

• A 15-amino acid tag-peptide was identified at the C-termini of trans-translation products in B. subtilis.
• The same tag-peptide was observed when B. subtilis tmRNA was coexpressed with the truncated gene in E. coli.
• Bacillus subtilis tmRNA demonstrated functionality, albeit at lower efficiency, in an in vitro E. coli system.

Conclusions:

• Bacillus subtilis tmRNA is functional in trans-translation, producing tagged polypeptides even in the absence of ribosomal protein S1.
• The tag-initiation mechanism on tmRNA appears conserved between Gram-positive and Gram-negative bacteria.
• These findings contribute to understanding bacterial protein quality control mechanisms across different species.