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Updated: Sep 10, 2026

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria
Published on: February 23, 2021
Long-range mRNA folding shapes expression and sequence of bacterial genes
Manraj S Gill1, Isabelle A Kim1, James R Xue2
1Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Abstract:
Bacterial gene expression is strongly influenced by local mRNA secondary structure, yet the impact of long-range folding remains poorly understood. Here, we show that sequences hundreds of nucleotides from the mRNA 5' end can act as potent repressors of gene expression through long-range base pairing to the ribosome binding site (RBS), subjecting anti-RBS sequences to negative selection. Using massively parallel reporter assays in Bacillus subtilis, we identify anti-RBS sequences as among the strongest determinants of reduced mRNA abundance across the transcript body. We demonstrate that distal anti-RBS elements engage in long-range folding with the Shine-Dalgarno sequence, blocking ribosome entry and promoting mRNA decay. Consistent with these repressive effects, anti-RBS-like sequences are depleted throughout diverse bacterial coding sequences but not from leaderless transcripts, and introducing distal anti-RBS to native genes reduces expression. Our findings establish that long-range mRNA folding is a conserved force shaping gene expression and constrains coding sequence evolution.
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