The internal ribosome entry site (IRES) in the hepatitis C virus (HCV) RNA genome is essential for the initiation of viral protein synthesis. IRES domains adopt well-defined folds that are potential targets for antiviral translation inhibitors. We have determined the three-dimensional structure of the IRES subdomain IIa in complex with a benzimidazole translation inhibitor at 2.2 Å resolution.Comparison to the structure of the unbound RNA in conjunction with studies of inhibitor binding to the target in solution demonstrate that the RNA undergoes a dramatic ligand-induced conformational adaptation to form a deep pocket that resembles the substrate binding sites in riboswitches. The presence of a well-defined ligand-binding pocket within the highly conserved IRES subdomain IIa holds promise for the development of unique anti-HCV drugs with a high barrier to resistance.crystallography | hepatitis C virus inhibitor | RNA structure I nfection with hepatitis C virus (HCV), which affects over 170 million individuals worldwide, is a leading cause of liver failure and hepatocellular carcinoma (1). Until earlier this year, when two protease inhibitors were approved as the first direct antiviral drugs for the treatment of HCV infection (2), the standard anti-HCV therapy consisted of an immunostimulatory regimen of pegylated interferon-α and the nucleoside analog ribavirin, which suffered from low efficacy as well as serious side effects (3). The prevalence of preexisting drug-resistance mutations in HCV quasispecies due to the low fidelity of the viral RNA-dependent RNA polymerase (NS5B) creates an urgent need for combination therapy with unique antiviral agents directed at distinct HCV targets (4).Among the potential targets for HCV inhibitors, the 5′ untranslated region (UTR) of the viral RNA genome stands out for its high sequence conservation within virus clinical isolates (5), which exceeds the conservation of the HCV protein reading frames. The HCV 5′ UTR harbors an internal ribosome entry site (IRES) which recruits host cell 40S ribosomal subunits and ultimately initiates translation of virus proteins via a 5′ cap-independent mechanism (6, 7). The function of the IRES relies on a structured RNA element, which contains several independently folding domains (Fig. 1A) (8, 9). The three-dimensional structure of the subdomain IIa target was previously determined in our laboratory revealing an overall bent architecture around an RNA internal loop (Fig. 1B) (10), in agreement with NMR analyses of the full domain II (11) and cryoelectron microscopy studies of 13). The L-shaped conformation of subdomain IIa directs the apical hairpin loop IIb toward the ribosomal E site in proximity of the active site. Ribosomal association of domain II induces a conformational change in the 40S head and closes the mRNA binding cleft. Both, the correct positioning of the viral mRNA initiation codon as well as the joining of the ribosomal subunits to form functional 80S units depend critically on the L-shaped architecture of the domain II (7...

BackgroundThe concerted effects of changes in gene expression due to changes in the environment are ultimately reflected in the metabolome. Dynamics of metabolite concentrations under a certain condition can therefore give a description of the cellular state with a high degree of functional information. We used this potential to evaluate the metabolic status of two recombinant strains of Saccharomyces cerevisiae during anaerobic batch fermentation of a glucose/xylose mixture. Two isogenic strains were studied, differing only in the pathways used for xylose assimilation: the oxidoreductive pathway with xylose reductase (XR) and xylitol dehydrogenase (XDH) or the isomerization pathway with xylose isomerase (XI). The isogenic relationship between the two strains ascertains that the observed responses are a result of the particular xylose pathway and not due to unknown changes in regulatory systems. An increased understanding of the physiological state of these strains is important for further development of efficient pentose-utilizing strains for bioethanol production.ResultsUsing LC-MS/MS we determined the dynamics in the concentrations of intracellular metabolites in central carbon metabolism, nine amino acids, the purine nucleotides and redox cofactors. The general response to the transition from glucose to xylose was increased concentrations of amino acids and TCA-cycle intermediates, and decreased concentrations of sugar phosphates and redox cofactors. The two strains investigated had significantly different uptake rates of xylose which led to an enhanced response in the XI-strain. Despite the difference in xylose uptake rate, the adenylate energy charge remained high and stable around 0.8 in both strains. In contrast to the adenylate pool, large changes were observed in the guanylate pool.ConclusionsThe low uptake of xylose by the XI-strain led to several distinguished responses: depletion of key metabolites in glycolysis and NADPH, a reduced GTP/GDP ratio and accumulation of PEP and aromatic amino acids. These changes are strong indicators of carbon starvation. The XR/XDH-strain displayed few such traits. The coexistence of these traits and a stable adenylate charge indicates that xylose supplies energy to the cells but does not suppress a response similar to carbon starvation. Particular signals may play a role in the latter, of which the GTP/GMP ratio could be a candidate as it decreased significantly in both strains.

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Background The selection of bioengineering platform strains and engineering strategies to improve the stress resistance of Saccharomyces cerevisiae remains a pressing need in bio-based chemical production. Thus, a systematic effort to exploit genotypic and phenotypic diversity to boost yeast's industrial value is still urgently needed. Results We analyzed 5,400 growth curves obtained from 36 S. cerevisiae strains and comprehensively profiled their resistances against 13 industrially relevant stresses. We observed that bioethanol and brewing strains exhibit higher resistance against acidic conditions; however, plant isolates tend to have a wider range of resistance, which may be associated with their metabolome and fluxome signatures in the tricarboxylic acid cycle and fatty acid metabolism. By deep genomic sequencing, we found that industrial strains have more genomic duplications especially affecting transcription factors, showing that they result from disparate evolutionary paths in comparison with the environmental strains, which have more indels, gene deletions, and strain-specific genes. Genome-wide association studies coupled with protein-protein interaction networks uncovered novel genetic determinants of stress resistances. Conclusions These resistance-related engineering targets and strain rankings provide a valuable source for engineering significantly improved industrial platform strains.