Genome-wide screening reveals metabolic regulation of translational fidelity

Abstract

Translational quality control is critical for maintaining the accuracy of protein synthesis in all domains of life. Mutations in aminoacyl-tRNA synthetases and the ribosome are known to affect translational fidelity and alter fitness, viability, stress responses, neuron function, and life span. In this study, we used a high-throughput fluorescence-based assay to screen a knock-out library of Escherichia coli and identified 30 nonessential genes that are critical for maintaining the fidelity of stop-codon readthrough. Most of these identified genes have not been shown to affect translational fidelity previously. Intriguingly, we show that several genes controlling metabolism, including cyaA and guaA, unexpectedly enhance stop-codon readthrough. CyaA and GuaA catalyze the synthesis of cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (GMP), respectively. Both CyaA and GuaA increase the expression of ribosomes and tRNAs, allowing aminoacyl-tRNAs to compete with release factors and suppress stop codons. In addition, the effect of guaA deletion on stop-codon readthrough is abolished by deleting prfC, which encodes release factor 3 (RF3). Our results suggest that nucleotide and carbon metabolism is tightly coupled with translational fidelity.

Deficiency in ribosome biogenesis causes streptomycin resistance and impairs motility inSalmonella

“…Mutations in uS12 that lead to Str resistance often improve ribosomal fidelity, which counters the effect of Str to induce mistranslation (32,33). Deleting rsmG also increases ribosomal fidelity in E. coli and M. tuberculosis (27,34). Using a dual-fluorescence reporter that our lab previously developed (35,36), we found that the Salmonella ΔrsmG strain exhibited decreased readthrough of UGA stop codons (Fig.…”

Translational Fidelity during Bacterial Stresses and Host Interactions

“…The connection between metabolism and translational fidelity is further revealed by a recent genetic screening [ 73 ]. In a genome-wide screening of an E. coli knockout library, we have identified several genes that control metabolic processes to affect stop-codon readthrough.…”

“…In particular, CyaA controls the synthesis of cyclic AMP, which is a master regulator of metabolic pathways [ 74 ]. We show that deleting cyaA decreases readthrough of stop codons, at least partially by repressing the expression of tRNAs that compete with release factors [ 73 ]. It is possible that amino acid imbalance may also contribute to the efficiency of readthrough.…”

“…Transfer RNAs are heavily modified molecules, and modifications in the anticodon loop often perturb the accuracy of ribosomal decoding [ 83 , 84 ]. The mS2i6A37 modification is catalyzed by MiaABC and promotes stop-codon readthrough by near-cognate tRNAs [ 73 , 85 , 86 ]. In Salmonella , deletion of miaA induces pleiotropic effects on cell physiology, including decreased growth, altered sensitivity to several amino acids analogs, and hypersensitivity to oxidative and heat stresses [ 87 , 88 ].…”

“…GidB, which is in the same operon as GidA, is a methyltransferase responsible for N 7 methylation of G527 (m 7 G572) of the 16S ribosomal RNA [ 108 ]. GidB promotes UGA readthrough in E. coli [ 73 ]. Under nalidixic acid stress, the gidB deletion Salmonella mutant exhibits reduced motility, filamentous morphology, and smaller colony size compared to the WT [ 109 ].…”