RESUMEN
In bacteria, release of newly synthesized proteins from ribosomes during translation termination is catalyzed by class-I release factors (RFs) RF1 or RF2, reading UAA and UAG or UAA and UGA codons, respectively. Class-I RFs are recycled from the post-termination ribosome by a class-II RF, the GTPase RF3, which accelerates ribosome intersubunit rotation and class-I RF dissociation. How conformational states of the ribosome are coupled to the binding and dissociation of the RFs remains unclear and the importance of ribosome-catalyzed guanine nucleotide exchange on RF3 for RF3 recycling in vivo has been disputed. Here, we profile these molecular events using a single-molecule fluorescence assay to clarify the timings of RF3 binding and ribosome intersubunit rotation that trigger class-I RF dissociation, GTP hydrolysis, and RF3 dissociation. These findings in conjunction with quantitative modeling of intracellular termination flows reveal rapid ribosome-dependent guanine nucleotide exchange to be crucial for RF3 action in vivo.
Asunto(s)
Bacterias , Terminación de la Cadena Péptídica Traduccional , Factores de Terminación de Péptidos , Bacterias/metabolismo , Guanosina Trifosfato/metabolismo , Factores de Terminación de Péptidos/metabolismo , Unión ProteicaRESUMEN
Thermorubin (THB) is a long-known broad-spectrum ribosome-targeting antibiotic, but the molecular mechanism of its action was unclear. Here, our precise fast-kinetics assays in a reconstituted Escherichia coli translation system and 1.96 Å resolution cryo-EM structure of THB-bound 70S ribosome with mRNA and initiator tRNA, independently suggest that THB binding at the intersubunit bridge B2a near decoding center of the ribosome interferes with the binding of A-site substrates aminoacyl-tRNAs and class-I release factors, thereby inhibiting elongation and termination steps of bacterial translation. Furthermore, THB acts as an anti-dissociation agent that tethers the ribosomal subunits and blocks ribosome recycling, subsequently reducing the pool of active ribosomes. Our results show that THB does not inhibit translation initiation as proposed earlier and provide a complete mechanism of how THB perturbs bacterial protein synthesis. This in-depth characterization will hopefully spur efforts toward the design of THB analogs with improved solubility and effectivity against multidrug-resistant bacteria.
Asunto(s)
Subunidades Ribosómicas , Ribosomas , Bacterias , Antibacterianos/farmacología , Escherichia coli/genéticaRESUMEN
How aminoglycoside antibiotics limit bacterial growth and viability is not clearly understood. Here we employ fast kinetics to reveal the molecular mechanism of action of a clinically used, new-generation, semisynthetic aminoglycoside Arbekacin (ABK), which is designed to avoid enzyme-mediated deactivation common to other aminoglycosides. Our results portray complete picture of ABK inhibition of bacterial translation with precise quantitative characterizations. We find that ABK inhibits different steps of translation in nanomolar to micromolar concentrations by imparting pleotropic effects. ABK binding stalls elongating ribosomes to a state, which is unfavorable for EF-G binding. This prolongs individual translocation step from â¼50 ms to at least 2 s; the mean time of translocation increases inversely with EF-G concentration. ABK also inhibits translation termination by obstructing RF1/RF2 binding to the ribosome. Furthermore, ABK decreases accuracy of mRNA decoding (UUC vs. CUC) by â¼80 000 fold, causing aberrant protein production. Importantly, translocation and termination events cannot be completely stopped even with high ABK concentration. Extrapolating our kinetic model of ABK action, we postulate that aminoglycosides impose bacteriostatic effect mainly by inhibiting translocation, while they become bactericidal in combination with decoding errors.
Asunto(s)
Antibacterianos/farmacología , Dibekacina/análogos & derivados , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Ribosomas/efectos de los fármacos , Antibacterianos/química , Dibekacina/química , Dibekacina/farmacología , Cinética , Factor G de Elongación Peptídica/antagonistas & inhibidores , Factores de Terminación de Péptidos/antagonistas & inhibidores , Péptidos/metabolismo , Inhibidores de la Síntesis de la Proteína/química , ARN Mensajero/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/metabolismoRESUMEN
Ribosome profiling spectra bear rich information on translation control and dynamics. Yet, due to technical biases in library generation, extracting quantitative measures of discrete translation events has remained elusive. Using maximum likelihood statistics and data set from Escherichia coli we develop a robust method for neutralizing technical biases (e.g. base specific RNase preferences in ribosome-protected mRNA fragments (RPF) generation), which allows for correct estimation of translation times at single codon resolution. Furthermore, we validated the method with available datasets from E. coli treated with antibiotic to inhibit isoleucyl-tRNA synthetase, and two datasets from Saccharomyces cerevisiae treated with two RNases with distinct cleavage signatures. We demonstrate that our approach accounts for RNase cleavage preferences and provides bias-corrected translation times estimates. Our approach provides a solution to the long-standing problem of extracting reliable information about peptide elongation times from highly noisy and technically biased ribosome profiling spectra.
Asunto(s)
Extensión de la Cadena Peptídica de Translación , Ribosomas/metabolismo , Codón , Escherichia coli/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Modelos Genéticos , Ribonucleasas , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ARNRESUMEN
Microcin C (McC) is a peptide adenylate antibiotic produced by Escherichiacoli cells bearing a plasmid-borne mcc gene cluster. Most MccA precursors, encoded by validated mcc operons from diverse bacteria, are 7 amino acids long, but the significance of this precursor length conservation has remained unclear. Here, we created derivatives of E. colimcc operons encoding longer precursors and studied their synthesis and bioactivities. We found that increasing the precursor length to 11 amino acids and beyond strongly decreased antibiotic production. We found this decrease to depend on several parameters. First, reiterative synthesis of the MccA peptide by the ribosome was decreased at longer mccA open reading frames, leading to less efficient competition with other messenger RNAs. Second, the presence of a formyl group at the N-terminal methionine of the heptameric peptide had a strong stimulatory effect on adenylation by the MccB enzyme. No such formyl group stimulation was observed for longer peptides. Finally, the presence of the N-terminal formyl on the heptapeptide adenylate stimulated bioactivity, most likely at the uptake stage. Together, these factors should contribute to optimal activity of McC-like compounds as 7-amino-acid peptide moieties and suggest convergent evolution of several steps of the antibiotic biosynthesis pathway and their adjustment to sensitive cell uptake machinery to create a potent drug.IMPORTANCEEscherichia coli microcin C (McC) is a representative member of peptide-nucleotide antibiotics produced by diverse microorganisms. The vast majority of biosynthetic gene clusters responsible for McC-like compound production encode 7-amino-acid-long precursor peptides, which are C-terminally modified by dedicated biosynthetic enzymes with a nucleotide moiety to produce a bioactive compound. In contrast, the sequences of McC-like compound precursor peptides are not conserved. Here, we studied the consequences of E. coli McC precursor peptide length increase on antibiotic production and activity. We show that increasing the precursor peptide length strongly decreases McC production by affecting multiple biosynthetic steps, suggesting that the McC biosynthesis system has evolved under significant functional constraints to maintain the precursor peptide length.
Asunto(s)
Antibacterianos/metabolismo , Antibacterianos/farmacología , Bacteriocinas/metabolismo , Bacteriocinas/farmacología , Escherichia coli/metabolismo , Biosíntesis de Proteínas , Ribosomas/metabolismo , Bacteriocinas/genética , Análisis Mutacional de ADN , Escherichia coli/genética , N-Formilmetionina/metabolismo , Sistemas de Lectura Abierta , PlásmidosRESUMEN
Accurate translation of genetic information is crucial for synthesis of functional proteins in all organisms. We use recent experimental data to discuss how induced fit affects accuracy of initial codon selection on the ribosome by aminoacyl transfer RNA in ternary complex ( T3) with elongation factor Tu (EF-Tu) and guanosine-5'-triphosphate (GTP). We define actual accuracy ([Formula: see text]) of a particular protein synthesis system as its current accuracy and the effective selectivity ([Formula: see text]) as [Formula: see text] in the limit of zero ribosomal binding affinity for T3. Intrinsic selectivity ([Formula: see text]), defined as the upper thermodynamic limit of [Formula: see text], is determined by the free energy difference between near-cognate and cognate T3 in the pre-GTP hydrolysis state on the ribosome. [Formula: see text] is much larger than [Formula: see text], suggesting the possibility of a considerable increase in [Formula: see text] and [Formula: see text] at negligible kinetic cost. Induced fit increases [Formula: see text] and [Formula: see text] without affecting [Formula: see text], and aminoglycoside antibiotics reduce [Formula: see text] and [Formula: see text] at unaltered [Formula: see text].
Asunto(s)
Código Genético/genética , Ribosomas/química , HumanosRESUMEN
We studied the effects of aminoglycosides and changing Mg2+ ion concentration on the accuracy of initial codon selection by aminoacyl-tRNA in ternary complex with elongation factor Tu and GTP (T3) on mRNA programmed ribosomes. Aminoglycosides decrease the accuracy by changing the equilibrium constants of 'monitoring bases' A1492, A1493 and G530 in 16S rRNA in favor of their 'activated' state by large, aminoglycoside-specific factors, which are the same for cognate and near-cognate codons. Increasing Mg2+ concentration decreases the accuracy by slowing dissociation of T3 from its initial codon- and aminoglycoside-independent binding state on the ribosome. The distinct accuracy-corrupting mechanisms for aminoglycosides and Mg2+ ions prompted us to re-interpret previous biochemical experiments and functional implications of existing high resolution ribosome structures. We estimate the upper thermodynamic limit to the accuracy, the 'intrinsic selectivity' of the ribosome. We conclude that aminoglycosides do not alter the intrinsic selectivity but reduce the fraction of it that is expressed as the accuracy of initial selection. We suggest that induced fit increases the accuracy and speed of codon reading at unaltered intrinsic selectivity of the ribosome.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Código Genético , Magnesio/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Ribosomas/efectos de los fármacos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Cationes Bivalentes , Codón , Escherichia coli/genética , Escherichia coli/metabolismo , Gentamicinas/farmacología , Cinética , Neomicina/farmacología , Paromomicina/farmacología , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/metabolismo , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Fracciones Subcelulares/química , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
Initiation factor (IF) 2 controls the fidelity of translation initiation by selectively increasing the rate of 50S ribosomal subunit joining to 30S initiation complexes (ICs) that carry an N-formyl-methionyl-tRNA (fMet-tRNAfMet). Previous studies suggest that rapid 50S subunit joining involves a GTP- and fMet-tRNAfMet-dependent "activation" of IF2, but a lack of data on the structure and conformational dynamics of 30S IC-bound IF2 has precluded a mechanistic understanding of this process. Here, using an IF2-tRNA single-molecule fluorescence resonance energy transfer signal, we directly observe the conformational switch that is associated with IF2 activation within 30S ICs that lack IF3. Based on these results, we propose a model of IF2 activation that reveals how GTP, fMet-tRNAfMet, and specific structural elements of IF2 drive and regulate this conformational switch. Notably, we find that domain III of IF2 plays a pivotal, allosteric, role in IF2 activation, suggesting that this domain can be targeted for the development of novel antibiotics.
Asunto(s)
Escherichia coli/fisiología , Factor 2 Procariótico de Iniciación/fisiología , Biosíntesis de Proteínas/fisiología , Subunidades Ribosómicas Grandes Bacterianas/fisiología , Subunidades Ribosómicas Pequeñas Bacterianas/fisiología , Regulación Alostérica/fisiología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiología , Transferencia Resonante de Energía de Fluorescencia/métodos , Guanosina Trifosfato/metabolismo , Cinética , Modelos Biológicos , Mutación , Factor 2 Procariótico de Iniciación/química , Conformación Proteica , Dominios Proteicos/fisiología , ARN de Transferencia de Metionina/metabolismo , Imagen Individual de Molécula/métodosRESUMEN
Two sets of ribosome structures have recently led to two different interpretations of what limits the accuracy of codon translation by transfer RNAs. In this review, inspired by this intermezzo at the Ribosome Club, we briefly discuss accuracy amplification by energy driven proofreading and its implementation in genetic code translation. We further discuss general ways by which the monitoring bases of 16S rRNA may enhance the ultimate accuracy (d-values) and how the codon translation accuracy is reduced by the actions of Mg2+ ions and the presence of error inducing aminoglycoside antibiotics. We demonstrate that complete freezing-in of cognate-like tautomeric states of ribosome-bound nucleotide bases in transfer RNA or messenger RNA is not compatible with recent experiments on initial codon selection by transfer RNA in ternary complex with elongation factor Tu and GTP. From these considerations, we suggest that the sets of 30S subunit structures from the Ramakrishnan group and 70S structures from the Yusupov/Yusupova group may, after all, reflect two sides of the same coin and how the structurally based intermezzo at the Ribosome Club may be resolved simply by taking the dynamic aspects of ribosome function into account.This article is part of the themed issue 'Perspectives on the ribosome'.
Asunto(s)
Codón/metabolismo , Biosíntesis de Proteínas , ARN Ribosómico 16S/genética , ARN de Transferencia/química , Ribosomas/química , Bacterias/química , Bacterias/genética , Eucariontes/química , Eucariontes/genética , ARN Ribosómico 16S/químicaRESUMEN
The antibiotic drug fusidic acid (FA) is commonly used in the clinic against gram-positive bacterial infections. FA targets ribosome-bound elongation factor G (EF-G), a translational GTPase that accelerates both messenger RNA (mRNA) translocation and ribosome recycling. How FA inhibits translocation was recently clarified, but FA inhibition of ribosome recycling by EF-G and ribosome recycling factor (RRF) has remained obscure. Here we use fast kinetics techniques to estimate mean times of ribosome splitting and the stoichiometry of GTP hydrolysis by EF-G at varying concentrations of FA, EF-G and RRF. These mean times together with previous data on uninhibited ribosome recycling were used to clarify the mechanism of FA inhibition of ribosome splitting. The biochemical data on FA inhibition of translocation and recycling were used to model the growth inhibitory effect of FA on bacterial populations. We conclude that FA inhibition of translocation provides the dominant cause of bacterial growth reduction, but that FA inhibition of ribosome recycling may contribute significantly to FA-induced expression of short regulatory open reading frames, like those involved in FA resistance.
Asunto(s)
Antibacterianos/farmacología , Ácido Fusídico/farmacología , Factor G de Elongación Peptídica/antagonistas & inhibidores , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Ribosómicas/antagonistas & inhibidores , Ribosomas/efectos de los fármacos , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Guanosina Trifosfato/metabolismo , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Terminación de la Cadena Péptídica Traduccional/efectos de los fármacosRESUMEN
How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec(-1), is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of â¼5 sec(-1), are discussed in the perspective of rapidly growing bacterial cells.
Asunto(s)
Fenómenos Fisiológicos Bacterianos , Ribosomas/fisiología , Guanosina Trifosfato/metabolismo , Cinética , Ribosomas/metabolismoRESUMEN
We have studied the pH dependence of the rate of termination of bacterial protein synthesis catalyzed by a class-1 release factor (RF1 or RF2). We used a classical quench-flow technique and a newly developed stopped-flow technique that relies on the use of fluorescently labeled peptides. We found the termination rate to increase with increasing pH and, eventually, to saturate at about 70 s(-1) with an apparent pKa value of about 7.6. From our data, we suggest that class-1 RF termination is rate limited by the chemistry of ester bond hydrolysis at low pH and by a stop-codon-dependent and pH-independent conformational change of RFs at high pH. We propose that RF-dependent termination depends on the participation of a hydroxide ion rather than a water molecule in the hydrolysis of the ester bond between the P-site tRNA and its peptide chain. We provide a simple explanation for why the rate of termination saturated at high pH in our experiments but not in those of others.
Asunto(s)
Proteínas Bacterianas/metabolismo , Codón de Terminación/metabolismo , Factores de Terminación de Péptidos/metabolismo , Biosíntesis de Proteínas/fisiología , ARN Mensajero/genética , Ribosomas/metabolismo , Codón de Terminación/genética , Concentración de Iones de Hidrógeno , Modelos Moleculares , Fragmentos de Péptidos/metabolismo , Factores de Terminación de Péptidos/genética , ARN Mensajero/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/genéticaRESUMEN
The antibiotic fusidic acid (FA) targets elongation factor G (EF-G) and inhibits ribosomal peptide elongation and ribosome recycling, but deeper mechanistic aspects of FA action have remained unknown. Using quench flow and stopped flow experiments in a biochemical system for protein synthesis and taking advantage of separate time scales for inhibited (10 s) and uninhibited (100 ms) elongation cycles, a detailed kinetic model of FA action was obtained. FA targets EF-G at an early stage in the translocation process (I), which proceeds unhindered by the presence of the drug to a later stage (II), where the ribosome stalls. Stalling may also occur at a third stage of translocation (III), just before release of EF-G from the post-translocation ribosome. We show that FA is a strong elongation inhibitor (K50% ≈ 1 µm), discuss the identity of the FA targeted states, and place existing cryo-EM and crystal structures in their functional context.
Asunto(s)
Antibacterianos/farmacología , Ácido Fusídico/farmacología , Factor G de Elongación Peptídica/antagonistas & inhibidores , Inhibidores de la Síntesis de la Proteína/farmacología , Relación Dosis-Respuesta a Droga , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Factor G de Elongación Peptídica/metabolismo , Ribosomas/metabolismoRESUMEN
Applications of N-methyl amino acids (NMAAs) in drug discovery are limited by their low efficiencies of ribosomal incorporation, and little is known mechanistically about the steps leading to incorporation. Here, we demonstrate that a synthetic tRNA body based on a natural N-alkyl amino acid carrier, tRNA(Pro), increases translation incorporation rates of all three studied NMAAs compared with tRNA(Phe)- and tRNA(Ala)-based bodies. We also investigate the pH dependence of the incorporation rates and find that the rates increase dramatically in the range of pH 7 to 8.5 with the titration of a single proton. Results support a rate-limiting peptidyl transfer step dependent on deprotonation of the N-nucleophile of the NMAA. Competition experiments demonstrate that several futile cycles of delivery and rejection of A-site NMAA-tRNA are required per peptide bond formed and that increasing magnesium ion concentration increases incorporation yield. Data clarify the mechanism of ribosomal NMAA incorporation and provide three generalizable ways to improve incorporation of NMAAs in translation.
Asunto(s)
Aminoácidos/metabolismo , Escherichia coli/metabolismo , Biosíntesis de Péptidos/fisiología , ARN Mensajero/genética , ARN de Transferencia de Prolina/metabolismo , Ribosomas/metabolismo , Escherichia coli/genética , Concentración de Iones de Hidrógeno , Cinética , Modelos Biológicos , Estructura MolecularRESUMEN
There is evidence that tRNA bodies have evolved to reduce differences between aminoacyl-tRNAs in their affinity to EF-Tu. Here, we study the kinetics of incorporation of L-amino acids (AAs) Phe, Ala allyl-glycine (aG), methyl-serine (mS), and biotinyl-lysine (bK) using a tRNA(Ala)-based body (tRNA(AlaB)) with a high affinity for EF-Tu. Results are compared with previous data on the kinetics of incorporation of the same AAs using a tRNA(PheB) body with a comparatively low affinity for EF-Tu. All incorporations exhibited fast and slow phases, reflecting the equilibrium fraction of AA-tRNA in active ternary complex with EF-Tu:GTP before the incorporation reaction. Increasing the concentration of EF-Tu increased the amplitude of the fast phase and left its rate unaltered. This allowed estimation of the affinity of each AA-tRNA to EF-Tu:GTP during translation, showing about a 10-fold higher EF-Tu affinity for AA-tRNAs formed from the tRNA(AlaB) body than from the tRNA(PheB) body. At â¼1 µM EF-Tu, tRNA(AlaB) conferred considerably faster incorporation kinetics than tRNA(PheB), especially in the case of the bulky bK. In contrast, the swap to the tRNA(AlaB) body did not increase the fast phase fraction of N-methyl-Phe incorporation, suggesting that the slow incorporation of N-methyl-Phe had a different cause than low EF-Tu:GTP affinity. The total time for AA-tRNA release from EF-Tu:GDP, accommodation, and peptidyl transfer on the ribosome was similar for the tRNA(AlaB) and tRNA(PheB) bodies. We conclude that a tRNA body with high EF-Tu affinity can greatly improve incorporation of unnatural AAs in a potentially generalizable manner.
Asunto(s)
Escherichia coli/genética , Factor Tu de Elongación Peptídica/genética , Aminoacil-ARN de Transferencia/genética , Ribosomas/genética , Aminoácidos/genética , Guanosina Trifosfato/genética , Cinética , Biosíntesis de ProteínasRESUMEN
OBJECTIVE: To investigate whether a diagnosis of anxiety disorder is a risk factor for adverse obstetric and neonatal outcome. METHODS: A retrospective population-based study was conducted comparing obstetric and neonatal complications in patients with and without a diagnosis of anxiety. Multivariable analysis was performed to control for confounders. RESULTS: During the study period 256,312 singleton deliveries have occurred, out of which 224 (0.09%) were in patients with a diagnosis of an anxiety disorder. Patients with anxiety disorders were older (32.17 ± 5.1 versus 28.56 ± 5.9), were more likely to be smokers (7.1% versus 1.1%) and had a higher rate of preterm deliveries (PTD; 15.2% versus 7.9%), as compared with the comparison group. Using a multiple logistic regression model, anxiety disorders were independently associated with advanced maternal age (OR 1.087; 95% CI 1.06-1.11; p = 0.001), smoking (OR 4.51; 95% CI 2.6-7.29; p = 0.001) and preterm labor (OR 1.92; 95% CI 1.32--2.8; p = 0.001). In addition, having a diagnosis of an anxiety disorder was found to be an independent risk factor for cesarean section (adjusted OR 2.5; 95% CI 1.82-3.46; p < 0.001), using another multivariable model. No association was noted between anxiety disorders and adverse neonatal outcomes including small for gestational age, low Apgar scores and perinatal mortality. CONCLUSION: Anxiety disorders are independent risk factors for spontaneous preterm delivery and cesarean section, but in our population it is not associated with adverse perinatal outcome.
Asunto(s)
Trastornos de Ansiedad/complicaciones , Complicaciones del Trabajo de Parto/etiología , Adulto , Trastornos de Ansiedad/epidemiología , Femenino , Humanos , Recién Nacido , Israel/epidemiología , Complicaciones del Trabajo de Parto/epidemiología , Embarazo , Estudios Retrospectivos , Adulto JovenRESUMEN
Bacterial populations growing in a changing world must adjust their proteome composition in response to alterations in the environment. Rapid proteome responses to growth medium changes are expected to increase the average growth rate and fitness value of these populations. Little is known about the dynamics of proteome change, e.g., whether bacteria use optimal strategies of gene expression for rapid proteome adjustments and if there are lower bounds to the time of proteome adaptation in response to growth medium changes. To begin answering these types of questions, we modeled growing bacteria as stoichiometrically coupled networks of metabolic pathways. These are balanced during steady-state growth in a constant environment but are initially unbalanced after rapid medium shifts due to a shortage of enzymes required at higher concentrations in the new environment. We identified an optimal strategy for rapid proteome adjustment in the absence of protein degradation and found a lower bound to the time of proteome adaptation after medium shifts. This minimal time is determined by the ratio between the Kullback-Leibler distance from the pre- to the postshift proteome and the postshift steady-state growth rate. The dynamics of optimally controlled proteome adaptation has a simple analytical solution. We used detailed numerical modeling to demonstrate that realistic bacterial control systems can emulate this optimal strategy for rapid proteome adaptation. Our results may provide a conceptual link between the physiology and population genetics of growing bacteria.
Asunto(s)
Adaptación Biológica/fisiología , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Modelos Biológicos , Proteoma/metabolismo , Proteómica/métodosRESUMEN
Translations with unnatural amino acids (AAs) are generally inefficient, and kinetic studies of their incorporations from transfer ribonucleic acids (tRNAs) are few. Here, the incorporations of small and large, non-N-alkylated, unnatural l-AAs into dipeptides were compared with those of natural AAs using quench-flow techniques. Surprisingly, all incorporations occurred in two phases: fast then slow, and the incorporations of unnatural AA-tRNAs proceeded with rates of fast and slow phases similar to those for natural Phe-tRNA(Phe). The slow phases were much more pronounced with unnatural AA-tRNAs, correlating with their known inefficient incorporations. Importantly, even for unnatural AA-tRNAs the fast phases could be made dominant by using high EF-Tu concentrations and/or lower reaction temperature, which may be generally useful for improving incorporations. Also, our observed effects of EF-Tu concentration on the fraction of the fast phase of incorporation enabled direct assay of the affinities of the AA-tRNAs for EF-Tu during translation. Our unmodified tRNA(Phe) derivative adaptor charged with a large unnatural AA, biotinyl-lysine, had a very low affinity for EF-Tu:GTP, while the small unnatural AAs on the same tRNA body had essentially the same affinities to EF-Tu:GTP as natural AAs on this tRNA, but still 2-fold less than natural Phe-tRNA(Phe). We conclude that the inefficiencies of unnatural AA-tRNA incorporations were caused by inefficient delivery to the ribosome by EF-Tu, not slow peptide bond formation on the ribosome.
Asunto(s)
Péptidos/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Cinética , Péptidos/síntesis química , Péptidos/química , Aminoacil-ARN de Transferencia/químicaRESUMEN
Enzyme inhibitors are used in many areas of the life sciences, ranging from basic research to the combat of disease in the clinic. Inhibitors are traditionally characterized by how they affect the steady-state kinetics of enzymes, commonly analyzed on the assumption that enzyme-bound and free substrate molecules are in equilibrium. This assumption, implying that an enzyme-bound substrate molecule has near zero probability to form a product rather than dissociate, is valid only for very inefficient enzymes. When it is relaxed, more complex but also more information-rich steady-state kinetics emerges. Although solutions to the general steady-state kinetics problem exist, they are opaque and have been of limited help to experimentalists. Here we reformulate the steady-state kinetics of enzyme inhibition in terms of new parameters. These allow for assessment of ambiguities of interpretation due to kinetic scheme degeneracy and provide an intuitively simple way to analyze experimental data. We illustrate the method by concrete examples of how to assess scheme degeneracy and obtain experimental estimates of all available rate and equilibrium constants. We suggest simple, complementary experiments that can remove ambiguities and greatly enhance the accuracy of parameter estimation.
Asunto(s)
Inhibidores Enzimáticos/química , Enzimas/química , Enzimas/metabolismo , Cinética , Especificidad por Sustrato , TermodinámicaRESUMEN
We previously identified mutations in the GTPase initiation factor 2 (IF2), located outside its tRNA-binding domain, compensating strongly (A-type) or weakly (B-type) for initiator tRNA formylation deficiency. We show here that rapid docking of 30S with 50S subunits in initiation of translation depends on switching 30S subunit-bound IF2 from its inactive to active form. Activation of wild-type IF2 requires GTP and formylated initiator tRNA (fMet-tRNA(i)). In contrast, extensive activation of A-type IF2 occurs with only GTP or with GDP and fMet-tRNA(i), implying a passive role for initiator tRNA as activator of IF2 in subunit docking. The theory of conditional switching of GTPases quantitatively accounts for all our experimental data. We find that GTP, GDP, fMet-tRNA(i) and A-type mutations multiplicatively increase the equilibrium ratio, K, between active and inactive forms of IF2 from a value of 4 × 10(-4) for wild-type apo-IF2 by factors of 300, 8, 80 and 20, respectively. Functional characterization of the A-type mutations provides keys to structural interpretation of conditional switching of IF2 and other multidomain GTPases.