ABC-F proteins have evaded functional characterization even though they comprise one of the most widely distributed branches of the ATP-binding cassette (ABC) superfamily. Herein, we demonstrate that YjjK, the most prevalent eubacterial ABC-F protein, gates ribosome entry into the translation elongation cycle through a nucleotide-dependent interaction sensitive to ATP/ADP ratio. Accordingly, we rename this protein Energy-dependent Translational Throttle A (EttA). We determined the crystal structure of Escherichia coli EttA and used it to design mutants for biochemical studies, including enzymological assays of the initial steps of protein synthesis. These studies suggest that EttA may regulate protein synthesis in energy-depleted cells, which have a low ATP/ADP ratio. Consistent with this inference, ΔettA cells exhibit a severe fitness defect in long-term stationary phase. These studies demonstrate that an ABC-F protein regulates protein synthesis via a novel mechanism sensitive to cellular energy status.

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Protein synthesis is a highly conserved process in all living cells involving several members of the translation factor (TRAFAC) class of P-loop GTPases, which play essential roles during translation. The universally conserved GTPase HflX has previously been shown to associate with the 50S ribosomal subunit, as well as to bind and hydrolyze both GTP and ATP. In an effort to elucidate the cellular function of HflX, we have determined the kinetic parameters governing the interaction between HflX and these two purine nucleotides using fluorescence-based steady-state and pre-steady-state techniques. On the basis of these, we demonstrate that the GTPase and ATPase activity of HflX is stimulated by 50S and 70S ribosomal particles. However, given cellular concentrations of the two purine nucleotides, approximately 80% of HflX will be bound to guanine nucleotides, indicating that HflX may function as a guanine nucleotide dependent enzyme in vivo. Using a highly purified reconstituted in vitro translation system, we show that the GTPase activity of HflX is also stimulated by poly(U) programmed 70S ribosomes and that the ribosome-dependent GTPase stimulation is specifically inhibited by the antibiotic chloramphenicol, which binds to the large ribosomal subunit, but not by kanamycin, an aminoglycoside targeting the small ribosomal subunit.

Using a combination of biochemical, structural probing and rapid kinetics techniques we reveal for the first time that the universally conserved translational GTPase (trGTPase) HflX binds to the E-site of the 70S ribosome and that its GTPase activity is modulated by peptidyl transferase centre (PTC) and peptide exit tunnel (PET) binding antibiotics, suggesting a previously undescribed mode of action for these antibiotics. Our rapid kinetics studies reveal that HflX functions as a ribosome splitting factor that disassembles the 70S ribosomes into its subunits in a nucleotide dependent manner. Furthermore, our probing and hydrolysis studies show that the ribosome is able to activate trGTPases bound to its E-site. This is, to our knowledge, the first case in which the hydrolytic activity of a translational GTPase is not activated by the GTPase activating centre (GAC) in the ribosomal A-site. Furthermore, we provide evidence that the bound state of the PTC is able to regulate the GTPase activity of E-site bound HflX.

A comprehensive understanding of plant metabolism could provide a direct mechanism for improving nitrogen use efficiency (NUE) in crops. One of the major barriers to achieving this outcome is our poor understanding of the complex metabolic networks, physiological factors, and signaling mechanisms that affect NUE in agricultural settings. However, an exciting collection of computational and experimental approaches has begun to elucidate whole-plant nitrogen usage and provides an avenue for connecting nitrogen-related phenotypes to genes. Herein, we describe how metabolomics, computational models of metabolism, and flux balance analysis have been harnessed to advance our understanding of plant nitrogen metabolism. We introduce a model describing the complex flow of nitrogen through crops in a real-world agricultural setting and describe how experimental metabolomics data, such as isotope labeling rates and analyses of nutrient uptake, can be used to refine these models. In summary, the metabolomics/computational approach offers an exciting mechanism for understanding NUE that may ultimately lead to more effective crop management and engineered plants with higher yields.

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