Molecular crowding limits translation and cell growth

Abstract

Bacterial growth is crucially dependent on protein synthesis and thus on the cellular abundance of ribosomes and related proteins. Here, we show that the slow diffusion of the bulky tRNA complexes in the crowded cytoplasm imposes a physical limit on the speed of translation, which ultimately limits the rate of cell growth. To study the required allocation of ancillary translational proteins to alleviate the effect of molecular crowding, we develop a model for cell growth based on a coarse-grained partitioning of the proteome. We find that coregulation of ribosome-and tRNA-affiliated proteins is consistent with measured growth-rate dependencies and results in near-optimal allocation over a broad range of growth rates. The analysis further resolves a long-standing controversy in bacterial growth physiology concerning the growth-rate dependence of translation speed and serves as a caution against premature identification of phenomenological parameters with mechanistic processes. Bacterial cell growth and protein synthesis are tightly coupled as proteins account for a large fraction of the cellular biomass (1). In the model organism Escherichia coli, over half of the biomass is protein (2), and protein synthesis accounts for more than two-thirds of the cell's ATP budget during rapid growth (3). Therefore, the machinery of protein synthesis, i.e., ribosomes, tRNAs, and ribosome-affiliated factors, plays a central role in maintaining exponential growth (1, 4). This is manifested by an increased ribosome content in rapidly growing cells (2,5,6), by direct observations that protein synthesis is limited by the availability of free ribosomes (7), and by considerations that link evolutionary selective pressure to the cost of protein synthesis (8).The most striking evidence for the central role of ribosomes in cell growth is provided by the linear relation between the ribosome mass fraction and the growth rate for bacteria grown in media containing different nutrients. This linear relation, which emerged from the systematic characterization of bacterial cells growing at different rates (5, 9), is illustrated in Fig. 1A with data for E. coli (2, 10, 11). It can be interpreted as reflecting the intrinsically autocatalytic activity of ribosomes synthesizing ribosomal proteins (9, 12) and identifies the fraction of ribosomes allocated to making ribosomal proteins as a key determinant of the growth rate (11). The picture that emerges from such considerations has formed the basis of a systematic theory of bacterial growth, based on empirical "growth laws", similar to the phenomenological laws of physics (11,13). The theory provides a successful framework for the analysis of the interdependence of cell growth and gene expression, of the effects of antibiotics, and of protein overexpression (11) without the need to characterize how the individual steps of synthesis and degradation are affected by the global state of the cell (14).In addition to their high ribosome content, rapidly growing bacteria also contain large amounts of othe...

Transfer RNA instability as a stress response inEscherichia coli: Rapid dynamics of the tRNA pool as a function of demand

“…Thus, we propose that the levels of components of the translational apparatus are not only co-regulated at the level of their synthesis, 5,9,93,96,97 but that the level of actively translating ribosomes indirectly regulates the tRNA pool, via degradation of the unengaged tRNAs. Importantly, to maintain a reasonable translation elongation rate even at very low concentrations of active ribosomes, we propose that their association with other protein factors, in particular the abundant elongation factor Tu, would protect a sizable fraction of the tRNA and thereby set a lower bound on the cellular concentration of ternary complexes.…”

“…92 and references within ref. 93). We hypothesize that the resolution of this apparent conflict lies in the realization that tRNA would not only be protected by active ribosomes, but most likely, to some degree, also by its other protein interaction partners, in particular elongation factor Tu.…”

“…In support of this hypothesis, the number of EF-Tu's per ribosome were shown to increase from ∼6 during rapid growth to ∼9 at very slow growth rates, thereby reaching a 1:1 stoichiometry with tRNA (ref. 93 and references therein). As we have demonstrated, a sudden amino acid starvation causes a substantial drop in tRNA concentrations, 14 and this situation differs from the growth at very low growth rates where a steady state has been reached over generations.…”

Biochemical reactions in crowded environments: revisiting the effects of volume exclusion with simulations

“…As a consequence, reactions are expected to be diffusion-limited or close to the diffusion limit. Specifically for ribosomes, it was recently proposed that the slow diffusion of ternary complexes (tRNAs charged with amino acids and GTP-activated elongation factor Tu) imposes a fundamental limitation on the speed of translation, which necessitates the large concentrations of elongation factors in rapidly growing bacteria [29] (elongation factor Tu is the most abundant protein in E. coli [30]). Such a limitation would be aggravated during growth under increased osmotic pressure.…”

Initiator tRNA Genes Template the 3’CCA End at High Frequencies in Bacteria

“…Under conditions of rapid growth, the cotranscriptional synthesis of 3′-terminal CCA in tRNAs can increase the allocation of cellular resources directly to the synthesis of new proteomic biomass and growth in two ways: first, by reducing steady-state cellular pools of species of nonfunctional tRNA precursors, which reduces the mass and energy overhead of the translational machinery itself, and second, by reducing the steady-state fraction of ribosomes devoted to synthesizing tRNA-affiliated proteins such as CCA-adding enzyme [28,29].…”