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

Accessibility of the ribosome binding site (RBS) plays an outsized role in bacterial mRNA decay and translation. Antagonistic mRNA sequences that reduce accessibility and regulate expression have been widely documented near the RBS. To determine whether such sequences are also the primary effectors of expression when placed far from the RBS, we measured impacts of all possible 8-nucleotide substitutions (65,536 variants) at different positions in mRNA in Bacillus subtilis. While the vast majority of substitutions negligibly affect RNA levels, pyrimidine-rich substitutions resembling the anti-Shine-Dalgarno (aSD) sequence exhibit strong inhibitory effects. Even several hundred nucleotides downstream of the RBS, these aSD-like sequences base-pair with the RBS, promote RNA decay, and inhibit translation initiation. We find aSD-like sequences to be depleted throughout endogenous genes, likely due to selective pressure for expression. Taken together, our findings reveal widespread long-range RNA intramolecular interactions in vivo and uncover a key constraint on gene sequence evolution.