In vivo tmRNA protection by SmpB and pre-ribosome binding conformation in solution

Area of Science:

• Bacterial molecular biology
• RNA structure and function
• Protein-RNA interactions

Background:

• Bacterial transfer-messenger RNA (tmRNA) is a unique molecule with both tRNA and mRNA characteristics.
• It plays a vital role in the trans-translation system, rescuing stalled ribosomes.
• The interaction with its partner protein, SmpB, is essential for tmRNA function.

Purpose of the Study:

• To investigate the in vivo binding of SmpB to tmRNA's tRNA-like domain (TLD).
• To understand how SmpB chaperones the folding of the TLD-H2 region of tmRNA.
• To elucidate the solution structure of tmRNA and its complex with SmpB before ribosome binding.

Main Methods:

• Utilizing a heterologous system to observe tmRNA degradation pathways.
• Employing mass spectrometry (MS) and nuclear magnetic resonance (NMR) for tmRNA digest analysis.
• Conducting co-overproduction assays of tmRNA and SmpB.
• Using small-angle X-ray scattering (SAXS) to study solution conformations.

Main Results:

• SmpB protein binds to the tRNA-like region (TLD) of tmRNA in vivo.
• SmpB effectively chaperones the folding of the TLD-H2 region.
• Magnesium ions (Mg2+) induce compaction of the tmRNA structure.
• SmpB also compacts tmRNA structure, though to a lesser extent than Mg2+.
• The intrinsic solution structure of tmRNA mirrors its topology when bound to ribosomes.

Conclusions:

• tmRNA possesses an intrinsic, stable structure in solution that is crucial for its function.
• This pre-existing structure facilitates efficient recruitment by the 70S ribosome.
• The SmpB-tmRNA interaction stabilizes the RNA structure, aiding the trans-translation process.