Long-range mRNA folding shapes expression and sequence of bacterial genes

Area of Science:

• Molecular Biology
• Bacterial Genetics
• RNA Biology

Background:

• Ribosome binding site (RBS) accessibility is crucial for bacterial mRNA translation and decay.
• Antagonistic mRNA sequences near the RBS regulate gene expression by limiting accessibility.

Purpose of the Study:

• To investigate the impact of mRNA sequence substitutions far from the RBS on gene expression.
• To determine if distant sequences can influence RNA stability and translation initiation.

Main Methods:

• Systematic analysis of all 65,536 possible 8-nucleotide substitutions at various mRNA positions in *Bacillus subtilis*.
• Measurement of RNA levels and translation efficiency for each variant.

Main Results:

• Pyrimidine-rich substitutions, similar to anti-Shine-Dalgarno (aSD) sequences, strongly inhibited expression.
• These aSD-like sequences, even hundreds of nucleotides downstream, base-paired with the RBS, increased RNA decay, and reduced translation initiation.
• Endogenous genes showed depletion of aSD-like sequences, suggesting evolutionary pressure against them.

Conclusions:

• Widespread long-range intramolecular RNA interactions occur *in vivo*.
• Distant RNA sequences can significantly impact gene expression by interacting with the RBS.
• The presence of aSD-like sequences imposes a constraint on bacterial gene sequence evolution.