Binding of 2′,3′-Cyclic Nucleotide Monophosphates to Bacterial Ribosomes Inhibits Translation

The intracellular small molecules 2′,3′-cyclic nucleotide monophosphates (2′,3′-cNMPs) have recently been rediscovered within both prokaryotes and eukaryotes. Studies in bacteria have demonstrated that 2′,3′-cNMP levels affect bacterial phenotypes, such as biofilm formation, motility, and growth, and modulate expression of numerous genes, suggesting that 2′,3′-cNMP levels are monitored by cells. In this study, 2′,3′-cNMP-linked affinity chromatography resins were used to identify Escherichia coli proteins that bind 2′,3′-cNMPs, with the top hits including all of the ribosomal proteins, and to confirm direct binding of purified ribosomes. Using in vitro translation assays, we have demonstrated that 2′,3′-cNMPs inhibit translation at concentrations found in amino acid-starved cells. In addition, a genetically encoded tool to increase cellular 2′,3′-cNMP levels was developed and was demonstrated to decrease E. coli growth rates. Taken together, this work suggests a mechanism for 2′,3-cNMP levels to modulate bacterial phenotypes by rapidly affecting translation.