Polypeptide-chain-elongation rate in Escherichia coli B/r as a function of growth rate

262

359

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...

Section: Coregulation Of Ribosomal and Trna-affiliated Proteins Correcontrasting

confidence: 41%

mentioning

confidence: 99%

Molecular crowding limits translation and cell growth

Klumpp

Scott

Pedersen

et al. 2013

262

359

“…3). We note that the minimal stalling time of ∼45 s after a successful viomycin attack is equivalent to the time required to translate roughly 900 codons in rapidly growing Escherichia coli cells with an average codon translation time of 50 ms (29). This greatly exceeds the average distance of 14-26 codons between ribosomes on mRNA (30), and therefore viomycin binding to a ribosome is expected to lead to queuing of trailing ribosomes behind it.…”

Section: Discussionmentioning

confidence: 99%

Molecular mechanism of viomycin inhibition of peptide elongation in bacteria

Holm

Borg

Ehrenberg

et al. 2016

Viomycin is a tuberactinomycin antibiotic essential for treating multidrug-resistant tuberculosis. It inhibits bacterial protein synthesis by blocking elongation factor G (EF-G) catalyzed translocation of messenger RNA on the ribosome. Here we have clarified the molecular aspects of viomycin inhibition of the elongating ribosome using pre-steadystate kinetics. We found that the probability of ribosome inhibition by viomycin depends on competition between viomycin and EF-G for binding to the pretranslocation ribosome, and that stable viomycin binding requires an A-site bound tRNA. Once bound, viomycin stalls the ribosome in a pretranslocation state for a minimum of ∼45 s. This stalling time increases linearly with viomycin concentration. Viomycin inhibition also promotes futile cycles of GTP hydrolysis by EF-G. Finally, we have constructed a kinetic model for viomycin inhibition of EF-G catalyzed translocation, allowing for testable predictions of tuberactinomycin action in vivo and facilitating in-depth understanding of resistance development against this important class of antibiotics.protein synthesis | viomycin | ribosome | antibiotics | tuberculosis T uberculosis (TB) is a global threat to human health, and over 9 million people contracted the disease in 2013 (1). The emergence of strains of Mycobacterium tuberculosis, the causative agent of TB, resistant to several first-and second-line antitubercular drugs is a significant concern. The tuberactinomycin antibiotics are bacterial protein synthesis inhibitors, commonly used as second-line drugs against multidrug-resistant TB. Viomycin was the first drug of this class to be discovered (2, 3). It is a cyclic pentapeptide that contains several nonstandard amino acids ( Fig. 1A) and is produced by a nonribosomal peptidyl transferase (4).Viomycin affects bacterial protein synthesis by inhibiting mRNA translocation (5) and causing misreading of the genetic code (6). The present study focuses on viomycin inhibition of translocation. During translocation the mRNA moves through the ribosome by one base triplet (codon), the peptidyl transfer RNA (tRNA) moves from the ribosomal A to P site, and the deacylated tRNA moves from the P to E site. Translocation is catalyzed by elongation factor G (EF-G) in a GTP hydrolysis-dependent manner (7) and occurs via formation of tRNA hybrid states (8), relative rotation of the ribosomal subunits (9), and movement of the L1 stalk (10). Bulk FRET, chemical footprinting, and single-molecule FRET experiments have shown that viomycin stabilizes the ribosome in a pretranslocation state with tRNAs in hybrid A/P and P/E configurations, rotated ribosomal subunits, and the L1 stalk in a closed conformation interacting with the P-site tRNA (11-16); this structure has been visualized by cryo-EM (17). However, a crystal structure of the viomycin-bound ribosome (18) and one single-molecule FRET study (19) have suggested stabilization of the tRNAs in the classical state.Recent crystal (18,20) and cryo-EM (17) structures of the viomycin-bound riboso...

“…Fig. 8 shows some rate distributions (for E. coli): protein folding, protein diffusion across the cell, rates of biochemical reactions (uncatalyzed and catalyzed), and rates of protein synthesis assuming a translation rate of 15 amino acids per second (70). The vertical bar on the right shows E. coli's roughly 20-min replication time under fast-growth conditions (1).…”

Section: Protein Folding Kineticsmentioning

confidence: 99%

Physical limits of cells and proteomes

Dill

Ghosh

Schmit

2011

281

280

What are the physical limits to cell behavior? Often, the physical limitations can be dominated by the proteome, the cell's complement of proteins. We combine known protein sizes, stabilities, and rates of folding and diffusion, with the known protein-length distributions PðNÞ of proteomes (Escherichia coli, yeast, and worm), to formulate distributions and scaling relationships in order to address questions of cell physics. Why do mesophilic cells die around 50°C? How can the maximal growth-rate temperature (around 37°C) occur so close to the cell-death temperature? The model shows that the cell's death temperature coincides with a denaturation catastrophe of its proteome. The reason cells can function so well just a few degrees below their death temperature is because proteome denaturation is so cooperative. Why are cells so dense-packed with protein molecules (about 20% by volume)? Cells are packed at a density that maximizes biochemical reaction rates. At lower densities, proteins collide too rarely. At higher densities, proteins diffuse too slowly through the crowded cell. What limits cell sizes and growth rates? Cell growth is limited by rates of protein synthesis, by the folding rates of its slowest proteins, and-for large cells-by the rates of its protein diffusion. Useful insights into cell physics may be obtainable from scaling laws that encapsulate information from protein knowledge bases. . So, some behaviors of cells are likely to be dominated by the physical properties of its proteomethe collection of its thousands of different types of proteins. We develop here some biophysical scaling relationships of proteomes, and we use those relationships to make estimates of the physical limits of cell behavior. Our scaling relationships come from combining current databases of the properties of proteins that have been measured in vitro, with PðNÞ, the length distributions of proteins that are known for several proteomes. Some of the hypotheses we explore are not new; what is previously undescribed is the use of modern databases to make quantitative estimates of physical limits. A key point, previously also made by Thirumalai (4), is that many physical properties of proteins just depend on N, the number of amino acids in the protein. We estimate the folding stabilities for mesophiles and thermophiles, the folding rates, and the diffusion coefficients of whole proteomes, and we compare these various rates at the end. We first consider the folding stabilities of proteomes. Proteomes Are Marginally Stable to DenaturationFor at least 116 monomeric, two-state and reversible folding proteins there are calorimetric measurements of the folding stability, ΔG ¼ G unfolded − G folded , enthalpy ΔH, entropy ΔS, and heat capacity, ΔC p . Data are now available for both mesophilic and thermophilic proteins. Taken over the full set of proteins, these thermal quantities depend, remarkably, mainly just on the number, N, of amino acids in the chain. The relationship is simply linear. For both enthalpy and entropy the correlati...