Translation during cold adaptation does not involve mRNA-rRNA base pairing through the downstream box

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• The downstream box (DB) is hypothesized to regulate mRNA translation, particularly for cold-shocked genes.
• A proposed mechanism involves base pairing between the DB and the anti-downstream box (antiDB) in 16S rRNA's helix 44.

Purpose of the Study:

• To investigate the controversial base-pairing interaction between the DB and antiDB during cold shock.
• To determine if this interaction is essential for cold acclimation and translation regulation in Escherichia coli.

Main Methods:

• Utilized an Escherichia coli strain with a modified helix 44 antiDB sequence, preventing mRNA base pairing.
• Assessed cold acclimation and cold shock survival.
• Compared translation efficiency of DB-containing and DB-lacking mRNAs in wild-type and mutant strains.
• Analyzed helix 44 structure using oligonucleotide binding and chemical probing (DMS, kethoxal).

Main Results:

• The modified strain exhibited normal cold acclimation and cold shock survival.
• Ribosomes from the mutant strain translated DB-containing and DB-lacking mRNAs with efficiencies comparable to the wild type.
• Structural analysis indicated helix 44 exists in a double-stranded conformation, unavailable for base pairing with mRNA under tested conditions.

Conclusions:

• The proposed DB-antiDB base-pairing interaction is unlikely to be the mechanism for DB function in cold shock.
• Helix 44's double-stranded structure prevents its direct interaction with mRNA sequences like the DB.
• Ribosomal function and cold acclimation are not dependent on this specific mRNA-rRNA interaction.