Characterizing the metabolic limitations of protein synthesis in model systems

Abstract

The regulation of protein synthesis is tailored to the metabolic state of the cell. This thesis explores the causes and consequences of restricted translation by ribosomes due to metabolic limitations in E. coli and mitochondria. First, systematic characterization of E. coli in balanced growth under carbon, nitrogen and phosphorus limitations at the same rate revealed three ways of tuning translation in response to nutrient limitation: carbon limitation slows translation by accumulating a large pool of inactive ribosomes; nitrogen limitation slows translation by slowing translational elongation; phosphorus limitation slows translation by reducing the total number of ribosomes. In addition to the growth-rate dependency of ribosome level as previously discovered, our study shows a surprising adaptation that phosphorous-limited E. coli cells can grow at the same rate as carbon- or nitrogen-limited cells with fewer ribosomes. Moreover, while manufacturing extra (unused) ribosomes can be energetically inefficient, the extra ribosomes accelerate growth when nutrients reappear. Second, examination of mitochondrial 1C metabolism unfolded a novel function of mitochondrial 5,10-methylenetetrahydrofolate for modifying mitochondrial tRNA at the wobble position. Missing the taurinomethyluridine base results in unstable codon-anticodon pairing and thus ribosome stalling on specific lysine (AAG) and leucine (UUG) codons. Together these results suggest an essential role of metabolism in regulating protein synthesis and provide new regulatory nodes in translation.