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

ABSTRACT: 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.

SUBMITTER: Chauhan SS

PROVIDER: S-EPMC9686202 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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

Chauhan Shikha S SS Marotta Nick J NJ Karls Anna C AC Weinert Emily E EE

ACS central science 20220921 11

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 <i>Escherichia ...[more]

PMID: 36439312

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Project description:Organismal adaptations to environmental stimuli are governed by intracellular signaling molecules such as nucleotide second messengers. Recent studies have identified functional roles for the noncanonical 2',3'-cyclic nucleotide monophosphates (2',3'-cNMPs) in both eukaryotes and prokaryotes. In Escherichia coli, 2',3'-cNMPs are produced by RNase I-catalyzed RNA degradation, and these cyclic nucleotides modulate biofilm formation through unknown mechanisms. The present work dissects cellular processes in E. coli and Salmonella enterica serovar Typhimurium that are modulated by 2',3'-cNMPs through the development of cell-permeable 2',3'-cNMP analogs and a 2',3'-cyclic nucleotide phosphodiesterase. Utilization of these chemical and enzymatic tools, in conjunction with phenotypic and transcriptomic investigations, identified pathways regulated by 2',3'-cNMPs, including flagellar motility and biofilm formation, and by oligoribonucleotides with 3'-terminal 2',3'-cyclic phosphates, including responses to cellular stress. Furthermore, interrogation of metabolomic and organismal databases has identified 2',3'-cNMPs in numerous organisms and homologs of the E. coli metabolic proteins that are involved in key eukaryotic pathways. Thus, the present work provides key insights into the roles of these understudied facets of nucleotide metabolism and signaling in prokaryotic physiology and suggest broad roles for 2',3'-cNMPs among bacteria and eukaryotes. IMPORTANCE Bacteria adapt to environmental challenges by producing intracellular signaling molecules that control downstream pathways and alter cellular processes for survival. Nucleotide second messengers serve to transduce extracellular signals and regulate a wide array of intracellular pathways. Recently, 2',3'-cyclic nucleotide monophosphates (2',3'-cNMPs) were identified as contributing to the regulation of cellular pathways in eukaryotes and prokaryotes. In this study, we define previously unknown cell processes that are affected by fluctuating 2',3'-cNMP levels or RNA oligomers with 2',3'-cyclic phosphate termini in E. coli and Salmonella Typhimurium, providing a framework for studying novel signaling networks in prokaryotes. Furthermore, we utilize metabolomics databases to identify additional prokaryotic and eukaryotic species that generate 2',3'-cNMPs as a resource for future studies.

Project description:Cyclic nucleotide-modulated ion channels play several essential physiological roles. They are involved in signal transduction in photoreceptors and olfactory sensory neurons as well as pacemaking activity in the heart and brain. Investigations of the molecular mechanism of their actions, including structural and electrophysiological characterization, are restricted by the availability of stable, purified protein obtained from accessible systems. Here, we establish that SthK, a cyclic nucleotide-gated (CNG) channel from Spirochaeta thermophila, is an excellent model for investigating the gating of eukaryotic CNG channels at the molecular level. The channel has high sequence similarity with its eukaryotic counterparts and was previously reported to be activated by cyclic nucleotides in patch-clamp experiments with Xenopus laevis oocytes. We optimized protein expression and purification to obtain large quantities of pure, homogeneous, and active recombinant SthK protein from Escherichia coli A negative-stain electron microscopy (EM) single-particle analysis indicated that this channel is a promising candidate for structural studies with cryo-EM. Using radioactivity and fluorescence flux assays, as well as single-channel recordings in lipid bilayers, we show that the protein is partially activated by micromolar concentrations of cyclic adenosine monophosphate (cAMP) and that channel activity is increased by depolarization. Unlike previous studies, we find that cyclic guanosine monophosphate (cGMP) is also able to activate SthK, but with much lower efficiency than cAMP. The distinct sensitivities to different ligands resemble eukaryotic CNG and hyperpolarization-activated and cyclic nucleotide-modulated channels. Using a fluorescence binding assay, we show that cGMP and cAMP bind to SthK with similar apparent affinities, suggesting that the large difference in channel activation by cAMP or cGMP is caused by the efficacy with which each ligand promotes the conformational changes toward the open state. We conclude that the functional characteristics of SthK reported here will permit future studies to analyze ligand gating and discrimination in CNG channels.

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

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

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