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Funding: This work was partly funded by the Perry foundation (JAP) (https://www.perryfoundation.co.uk/), The Rural and Environment Science and Analytical Services (RESAS) division of the Scottish Government (JAP & JTJ) (https://www.gov.scot/) and BBSRC award BB/V00249X/1 (JAP, TS & JTJ) (https://www.ukri.org/councils/bbsrc/). Host-specific plant pathogens must coordinate their life cycles with the availability of a host plant. Although this is frequently achieved through a response to specific chemical cues derived from the host plant, little is known about the molecular basis of the response to such cues and how these are used to trigger activation of the life cycle. In host-specific plant-parasitic cyst nematodes, unhatched juvenile nematodes lie dormant in the eggshell until chemical cues from a suitable host plant are detected and the hatching process is initiated. The molecular mechanisms by which hatch is linked to the presence of these chemical cues is unknown. We have identified a novel annexin-like protein that is localised to the eggshell of the potato cyst nematode Globodera rostochiensis. This annexin is unique in having a short peptide insertion that structural modelling predicts is present in one of the calcium-binding sites of this protein. Host-induced gene silencing of the annexin impacts the ability of the nematode to regulate and control permeability of the eggshell. We show that in the presence of the chemicals that induce hatching annexin lipid binding capabilities change, providing the first molecular link between a nematode eggshell protein and host-derived cues. This work demonstrates how a protein from a large family has been recruited to play a critical role in the perception of the presence of a host and provides a new potential route for control of cyst nematodes that impact global food production. Publisher PDF

Funding from the European Community's Seventh Framework Programme under grant agreements No. 602773 (Project KINDRED) was received for all partners in this work. This work also received funds from FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior through the Research Unit No. 4293 and project POCI-01-0145-FEDER-031013 (PTDC/SAUPAR/31013/2017 to NS); Individual funding from FCT through SFRH/BD/133485/2017 (to MS) and CEECIND/02362/2017 (to JT). Neglected tropical diseases caused by kinetoplastid parasites (Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp.) place a significant health and economic burden on developing nations worldwide. Current therapies are largely out-dated, inadequate and facing mounting drug resistance from the causative parasites. Thus, there is an urgent need for drug discovery and development. Target-led drug discovery approaches have focused on the identification of parasite enzymes catalysing essential biochemical processes, which significantly differ from equivalent proteins found in humans, thereby providing potentially exploitable therapeutic windows. One such target is ribose 5-phosphate isomerase B (RpiB), an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, which catalyses the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Although protozoan RpiB has been the focus of numerous targeted studies, compounds capable of selectively inhibiting this parasite enzyme have not been identified. Here, we present the results of a fragment library screening against Leishmania infantum RpiB, performed using thermal shift analysis. Hit fragments were shown to be effective inhibitors of LiRpiB in activity assays, and several were capable of selectively inhibiting parasite growth in vitro. These results support the identification of LiRpiB as a validated therapeutic target. The X-ray crystal structure of apo LiRpiB was also solved, permitting docking studies to assess how hit fragments might interact with LiRpiB to inhibit its activity. Overall, this work will guide structure-based development of LiRpiB inhibitors as anti-leishmanial agents. Postprint

KVGCS and SM thank DBT, New Delhi [BT/IN/Spain/39/SMl2017-18] for providing financial support. The financial assistance provided by DIST FIST grant (SR/FST/CSI-240/2012), New Delhi is gratefully acknowledged. SS thanks CSIR for providing SRF fellowship. Four groups, thirty-five compounds in total, of novel 1,2,3-triazole analogues of imidazo-[1,2-a]-pyridine-3-carboxamides were designed and synthesized using substituted pyridine, propargyl bromide, 2-azidoethyl 4-methyl benzenesulfonate and substituted acetylenes. These compounds were characterized using 1H NMR, 13C NMR, LCMS and elemental analyses and a crystal structure was obtained for one of the significantly active compounds, 8f. All the synthesized and characterized compounds were screened in vitro for antileishmanial and antitrypanosomal activity against Leishmania major and Trypanosoma brucei parasites, respectively. Among the tested analogues, fivecompounds (8d, 8f, 8j, 10b and 10d) exhibited significant antileishmanial activity while three compounds (10b, 11a and 11b) showed substantial activity against T. brucei parasite. In silico ADME prediction studies depicted that the essential compounds obeyed Lipinski's rule of five. The predicted in silico toxicity profile suggested that the tested compounds would be non-toxic, which was confirmed experimentally by the lack of cytotoxicity against HeLa cells. Finally, a molecular docking study was also performed, for 10d the most active antileishmanial compound, to study its putative binding pattern at the active site of the selected leishmanial trypanothione reductase target. Publisher PDF

It has long been established that mitochondrial dysfunction in Alzheimer&rsquo, s disease (AD) patients can trigger pathological changes in cell metabolism by altering metabolic enzymes such as the mitochondrial 17&beta, hydroxysteroid dehydrogenase type 10 (17&beta, HSD10), also known as amyloid-binding alcohol dehydrogenase (ABAD). We and others have shown that frentizole and riluzole derivatives can inhibit 17&beta, HSD10 and that this inhibition is beneficial and holds therapeutic merit for the treatment of AD. Here we evaluate several novel series based on benzothiazolylurea scaffold evaluating key structural and activity relationships required for the inhibition of 17&beta, HSD10. Results show that the most promising of these compounds have markedly increased potency on our previously published inhibitors, with the most promising exhibiting advantageous features like low cytotoxicity and target engagement in living cells.

myo -Inositol is a building block for all inositol-containing phospholipids in eukaryotes. It can be synthesized de novo from glucose-6-phosphate in the cytosol and endoplasmic reticulum. Alternatively, it can be taken up from the environment via Na + - or H + -linked myo -inositol transporters. While Na + -coupled myo -inositol transporters are found exclusively in the plasma membrane, H + -linked myo -inositol transporters are detected in intracellular organelles. In Trypanosoma brucei , the causative agent of human African sleeping sickness, myo -inositol metabolism is compartmentalized. De novo -synthesized myo -inositol is used for glycosylphosphatidylinositol production in the endoplasmic reticulum, whereas the myo -inositol taken up from the environment is used for bulk phosphatidylinositol synthesis in the Golgi complex. We now provide evidence that the Golgi complex-localized T. brucei H + -linked myo -inositol transporter (TbHMIT) is essential in bloodstream-form T. brucei . Downregulation of TbHMIT expression by RNA interference blocked phosphatidylinositol production and inhibited growth of parasites in culture. Characterization of the transporter in a heterologous expression system demonstrated a remarkable selectivity of TbHMIT for myo -inositol. It tolerates only a single modification on the inositol ring, such as the removal of a hydroxyl group or the inversion of stereochemistry at a single hydroxyl group relative to myo -inositol.

The striking feature of carbohydrates is their constitutional, conformational and configurational diversity. Biology has harnessed this diversity and manipulates carbohydrate residues in a variety of ways, one of which is epimerization. RmlC catalyzes the epimerization of the C3' and C5' positions of dTDP-6-deoxy-D-xylo-4-hexulose, forming dTDP-6-deoxy-L-lyxo-4-hexulose. RmlC is the third enzyme of the rhamnose pathway, and represents a validated anti-bacterial drug target. Although several structures of the enzyme have been reported, the mechanism and the nature of the intermediates have remained obscure. Despite its relatively small size (22 kDa), RmlC catalyzes four stereospecific proton transfers and the substrate undergoes a major conformational change during the course of the transformation. Here we report the structure of RmlC from several organisms in complex with product and product mimics. We have probed site-directed mutants by assay and by deuterium exchange. The combination of structural and biochemical data has allowed us to assign key residues and identify the conformation of the carbohydrate during turnover. Clear knowledge of the chemical structure of RmlC reaction intermediates may offer new opportunities for rational drug design.

Six diverse prokaryotic and five eukaryotic genomes were compared to deduce whether the protein synthesis termination signal has common determinants within and across both kingdoms. Four of the six prokaryotic and all of the eukaryotic genomes investigated demonstrated a similar pattern of nucleotide bias both 5' and 3' of the stop codon. A preferred core signal of 4 nt was evident, encompassing the stop codon and the following nucleotide. Codons decoded by hyper-modified tRNAs were over-represented in the region 5' to the stop codon in genes from both kingdoms. The origin of the 3' bias was more variable particularly among the prokaryotic organisms. In both kingdoms, genes with the highest expression index exhibited a strong bias but genes with the lowest expression showed none. Absence of bias in parasitic prokaryotes may reflect an absence of pressure to evolve more efficient translation. Experiments were undertaken to determine if a correlation existed between bias in signal abundance and termination efficiency. In Escherichia coli signal abundance correlated with termination efficiency for UAA and UGA stop codons, but not in mammalian cells. Termination signals that were highly inefficient could be made more efficient by increasing the concentration of the cognate decoding release factor.

Vancomycin, the last line of defense antibiotic, depends upon the attachment of the carbohydrate vancosamine to an aglycone skeleton for antibacterial activity. Vancomycin is a naturally occurring secondary metabolite that can be produced by bacterial fermentation. To combat emerging resistance, it has been proposed to genetically engineer bacteria to produce analogues of vancomycin. This requires a detailed understanding of the biochemical steps in the synthesis of vancomycin. Here we report the 1.4 A structure and biochemical characterization of EvaD, an RmlC-like protein that is required for the C-5' epimerization during synthesis of dTDP-epivancosamine. EvaD, although clearly belonging to the RmlC class of enzymes, displays very low activity in the archetypal RmlC reaction (double epimerization of dTDP-6-deoxy-4-keto-D-glucose at C-3' and C-5'). The high resolution structure of EvaD compared with the structures of authentic RmlC enzymes indicates that a subtle change in the enzyme active site repositions a key catalytic Tyr residue. A mutant designed to re-establish the normal position of the Tyr increases the RmlC-like activity of EvaD.

Bacteria have a rich collection of biochemical pathways for the synthesis of complex metabolites. These conversions often involve chemical reactions that are hard to reproduce in the laboratory. An area of considerable interest is in the manipulation and synthesis of carbohydrates. In contrast with amino acids, carbohydrates are densely functionalized (each carbon atom is attached to at least one heteroatom) and this holds out the prospect of discovering novel enzyme mechanisms. The results from the study of the biosynthetic dTDP-l-rhamnose pathway are discussed. dTDP-l-rhamnose is a key intermediate in many pathogenic bacteria, as it is the donor for l-rhamnose, which is found in the cell wall of important human pathogens, such as Mycobacteria tuberculosis and Salmonella typhimurium. All four enzymes have been structurally characterized; in particular, the acquisition of structural data on substrate complexes was extremely useful. The structural data have guided site-directed-mutagenesis studies that have been used to test mechanistic hypotheses. The results shed light on three classes of enzyme mechanism: nucleotide condensation, short-chain dehydrogenase activity and epimerization.

In this study we interrogate ~630 compounds of the Maybridge Rule of 3 Fragment Library for compounds that interact with, and inhibit TbCK. The Maybridge Rule of 3 Fragment Library is a small collection of quantifiable diverse, pharmacophoric rich, chemical entities that comply with the following criteria; MW ≤ 300, cLogP ≤ 3, H-Bond Acceptors ≤ 3, H-Bond Donors ≤ 3, Rotatable bonds (Flexibility Index) ≤ 3, Polar Surface Area ≤ 60 A2 and aqueous solubility ≥ 1 mM using LogS and high purity (≥ 95%). Comparisons between two different screening methods, a coupled enzyme activity assay and differential scanning fluorimetry, has allowed identification of compounds that interact and inhibit the T. brucei choline kinase, several of which possess selective trypanocidal activity. Screening of a comparatively small fragment library by two different screening methods has allowed identification of several compounds that interact with and inhibit TbCK, a genetically validated drug target against African sleeping sickness. Some of the inhibitory fragments were also selectively trypanocidal, considering these are relatively simple molecules with no optimization, finding low μΜ inhibitors is very encouraging. Moreover some of the morphological phenotypes of these trypanocidal compounds include cell-cycle arrests similar to those observed for the TbCK conditional knockout grown under permissive conditions.

Wide ranging studies of the readthrough of translational stop codons within the last 25 years have suggested that the stop codon might be only part of the molecular signature for recognition of the termination signal. Such studies do not distinguish between effects on suppression and effects on termination, and so we have used a number of different approaches to deduce whether the stop signal is a codon with a context or an extended factor recognition element. A data base of natural termination sites from a wide range of organisms (148 organisms, ∼40000 sequences) shows a very marked bias in the bases surrounding the stop codon in the genes for all organisms examined, with the most dramatic bias in the base following the codon (+4). The nature of this base determines the efficiency of the stop signal in vivo , and in Escherichia coli this is reinforced by overexpressing the stimulatory factor, release factor-3. Strong signals, defined by their high relative rates of selecting the decoding release factors, are enhanced whereas weak signals respond relatively poorly. Site-directed cross-linking from the +1, and bases up to +6 but not beyond make close contact with the bacterial release factor-2. The translational stop signal is deduced to be an extended factor recognition sequence with a core element, rather than simply a factor recognition triplet codon influenced by context.

The search for potent and selective sirtuin inhibitors continues as chemical tools of this type are of use in helping to assign the function of this interesting class of deacetylases. Here we describe SAR studies starting from the unselective sirtuin inhibitor tenovin-6. These studies identify a sub-micromolar inhibitor that has increased selectivity for SIRT2 over SIRT1 compared to tenovin-6. In addition, a H-1-NMR-based method is developed and used to validate further this class of sirtuin inhibitors. A thermal shift analysis of SIRT2 in the presence of tenovin-6, -43, a control tenovin and the known SIRT2 inhibitor AGK2 is also presented. Publisher PDF

Acivicin analogues with an increased affinity for CTP synthetase (CTPS) were designed as potential new trypanocidal agents. The inhibitory activity against CTPS can be improved by increasing molecular complexity, by inserting groups able to establish additional interactions with the binding pocket of the enzyme. This strategy has been pursued with the synthesis of α-amino-substituted analogues of Acivicin and N1-substituted pyrazoline derivatives. In general, there is direct correlation between the enzymatic activity and the invitro anti-trypanosomal efficacy of the derivatives studied here. However, this cannot be taken as a general rule, as other important factors may play a role, notably the ability of uptake/diffusion of the molecules into the trypanosomes. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inositol-3-phosphate synthase (INO1) has previously been genetically validated as a drug target againstTrypanosoma brucei, the causative agent of African sleeping sickness. Chemical intervention of this essential enzyme could lead to new therapeutic agents. Unfortunately, no potent inhibitors of INO1 from any organism have been reported, so a screen for potential novel inhibitors ofT. bruceiINO1was undertaken. Detection of inhibition ofT. bruceiINO1 is problematic due to the nature of the reaction. Direct detection requires differentiation between glucose-6-phosphate and inositol-3-phosphate. Coupled enzyme assays could give false positives as potentially they could inhibit the coupling enzyme. Thus, an alternative approach of differential scanning fluorimetry to identify compounds that interact withT. bruceiINO1 was employed to screen ~670 compounds from the MayBridge Rule of 3 Fragment Library. This approach identified 38 compounds, which significantly altered the Tmof TbINO1. Four compounds showed trypanocidal activity with ED50s in the tens of micromolar range, with 2 having a selectivity index in excess of 250. The trypanocidal and general cytotoxicity activities of all of the compounds in the library are also reported, with the best having ED50S of ~20 μM againstT. brucei.

The first convenient synthesis of enantiomerically pure (αS,5S)-α-amino-3-bromo-4,5-dihydroisoxazol-5-yl acetic acid (3-bromoacivicin) is described. We demonstrate that 3-bromoacivicin is a CTP synthetase inhibitor three times as potent as its 3-chloro analogue, the natural antibiotic acivicin. Because CTP synthetase was suggested to be a potential drug target in African trypanosomes, the in vitro/in vivo antitrypanosomal activity of 3-bromoacivicin was assessed in comparison with acivicin. Beyond expectation, we observed a 12-fold enhancement in the in vitro antitrypanosomal activity, while toxicity against mammalian cells remained unaffected. Despite its good in vitro activity and selectivity, 3-bromoacivicin proved to be trypanostatic and failed to completely eradicate the infection when tested in vivo at its maximum tolerable dose.

SUMMARYThe biological membranes ofTrypanosoma bruceicontain a complex array of phospholipids that are synthesizedde novofrom precursors obtained either directly from the host, or as catabolised endocytosed lipids. This paper describes the use of nanoflow electrospray tandem mass spectrometry and high resolution mass spectrometry in both positive and negative ion modes, allowing the identification of ~500 individual molecular phospholipids species from total lipid extracts of cultured bloodstream and procyclic formT. brucei. Various molecular species of all of the major subclasses of glycerophospholipids were identified including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol as well as phosphatidic acid, phosphatidylglycerol and cardolipin, and the sphingolipids sphingomyelin, inositol phosphoceramide and ethanolamine phosphoceramide. The lipidomic data obtained in this study will aid future biochemical phenotyping of either genetically or chemically manipulated commonly used bloodstream and procyclic strains ofTrypanosoma brucei. Hopefully this will allow a greater understanding of the bizarre world of lipids in this important human pathogen.

Originally published in: Protein Synthesis and Ribosome Structure. Edited by Knud H. Nierhaus and Daniel N. Wilson. Copyright © 2004 Wiley-VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3-527-30638-1 The sections in this article are Introduction Maintaining Decoding Accuracy and the Reading Frame The Use of a Stop Signal for both Elongation and Termination of Protein Synthesis The Mechanism for Sec Incorporation at UGA Sites in Bacterial mRNAs The Gene Products The Mechanism of Sec Incorporation The Competition between Sec Incorporation and Canonical Decoding of UGA by RF2 Mechanism for Sec Incorporation at UGA Sites in Eukaryotic and Archaeal mRNAs The Gene Products The Mechanism of Sec Incorporation at Specific UGA Stop Codons Why does Recoding Occur at Stop Signals? The Stop Signal of Prokaryotic Genomes — Engineered for High Efficiency Decoding? The Stop Signal of Eukaryotic Genomes – Diversity Contributes to Recoding Readthrough of a Stop Signal: Decoding Stop as Sense Bypassing of a Stop Codon: ‘Free-wheeling’ on the mRNA Frameshifting Around Stop or Sense Codons Forward Frameshifting: the +1 Event Programed −1 Frameshifting: A Common Mechanism used by Many Viruses During Gene Expression Conclusion Keywords: protein synthesis; ribosome structure; recoding in pro- and eukaryotes; stop signal for both elongation and termination of protein synthesis; Sec incorporation at UGA sites in bacterial mRNAs; Sec incorporation in eukaryotic and archaeal mRNAs; readthrough of a stop signal; bypassing of a stop codon; frameshifting around stop or sense codons

Molecular mimicry was a concept that was revived as we understood more about the ligands that bound to the active center of the ribosome, and the characteristics of the active center itself. It has been particularly useful for the termination phase of protein synthesis, because for many years this major process seemed not only to be out of step) with the initiation and elongation phases but also there were no common features of the process between eubacteria and eukaryotes. As the facts that supported molecular mimicry emerged, it was seen that the protein factors that facilitated polypeptide chain release when the decoding of an mRNA was complete had common features with the ligands involved in the other phases. Moreover, now common features and mechanisms began to emerge between the eubacterial and eukaryotic RFs and suddenly there seemed to be remarkable synergy between the external ligands and commonality in at least some features of the mechanistic prnciples. Almost 10 years after molecular mimicry took hold as a framework concept, we can now see that this idea is probably too simple. For example, structural mimicry can be apparent if there are extensive conformational changes either in the ribosome active center or in the ligand itself or, most likely, both. Early indications are that the bacterial RF may indeed undergo extensive conformational changes from its solution structure to achieve this accommodation. Thus, as important if not more important than structural and functional mimicry among the ligands, might be their accomodation of a common single active center made up of at least three parts to carry out a complex series of reactions. One part of the ribosomal active center is committed to decoding, a second is committed to the chemistry of putting the protein together and releasing it, and a third part, perhaps residing in the subdomains, is committed to binding ligands so that they can perform their respective single or multiple functions. It might be more accurate to regard the decoding RF as the cuckoo taking over the nest that was crafted and honed through evolution by another, the tRNA. A somewhat ungainly RF, perhaps bigger in dimensions than the tRNA, is able, nevertheless, like the cuckoo, to maneuvre into the nest. Perhaps it pushes the nest a little out of shape, but is still able to use the site for its own functions of stop signal decoding and for facilitating the release of the polypeptide. The term molecular mimicry has been dominant in the literature for a period of important advances in the understanding of protein synthesis. When the first structures of the ribosome appeared, the concept survived and was seen to be valid still. Now, we are at the stage of understanding the more detailed molecular interactions between ligands and the rRNA in particular, and how subtle changes in localized spatial orientations of atoms occur within these interactions. The simplicity of the original concept of mimicry will inevitably be blurred by this more detailed analysis. Nevertheless, it has provided a significant set of principles that allowed development of experimental programs to enhance our understanding of the dynamic events at this remarkable active site at the interface between the two subunits of this fascinating cell organelle, the ribosome.

Multi-layer perceptron neural networks that have been optimised by an evolutionary algorithm, were used to model experimentally determined protein synthesis termination signal strength. It is shown that the evolutionary algorithm produced much smaller multi-layer perceptrons with comparable or superior performance to manually designed networks.

Nature achieves the epimerization of carbohydrates by a variety of chemical routes. One common route is that performed by the class of enzyme defined by dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase (RmlC) from the rhamnose pathway. Earlier studies failed to identify the key residues in catalysis. We report the 1.3 A structure of RmlC from Streptococcus suis type 2 and its complexes with dTDP-D-glucose and dTDP-D-xylose. The streptococcal RmlC enzymes belong to a separate subgroup, sharing only 25% identity with RmlC from other bacteria, yet the S. suis enzyme has similar kinetic properties and structure to other RmlC enzymes. Structure, sequence alignment, and mutational analysis have now allowed reliable identification of the catalytic residues and their roles.

Molecular mimicry was a concept that was revived as we understood more about the ligands that bound to the active center of the ribosome, and the characteristics of the active center itself. It has been particularly useful for the termination phase of protein synthesis, because for many years this major process seemed not only to be out of step) with the initiation and elongation phases but also there were no common features of the process between eubacteria and eukaryotes. As the facts that supported molecular mimicry emerged, it was seen that the protein factors that facilitated polypeptide chain release when the decoding of an mRNA was complete had common features with the ligands involved in the other phases. Moreover, now common features and mechanisms began to emerge between the eubacterial and eukaryotic RFs and suddenly there seemed to be remarkable synergy between the external ligands and commonality in at least some features of the mechanistic prnciples. Almost 10 years after molecular mimicry took hold as a framework concept, we can now see that this idea is probably too simple. For example, structural mimicry can be apparent if there are extensive conformational changes either in the ribosome active center or in the ligand itself or, most likely, both. Early indications are that the bacterial RF may indeed undergo extensive conformational changes from its solution structure to achieve this accommodation. Thus, as important if not more important than structural and functional mimicry among the ligands, might be their accomodation of a common single active center made up of at least three parts to carry out a complex series of reactions. One part of the ribosomal active center is committed to decoding, a second is committed to the chemistry of putting the protein together and releasing it, and a third part, perhaps residing in the subdomains, is committed to binding ligands so that they can perform their respective single or multiple functions. It might be more accurate to regard the decoding RF as the cuckoo taking over the nest that was crafted and honed through evolution by another, the tRNA. A somewhat ungainly RF, perhaps bigger in dimensions than the tRNA, is able, nevertheless, like the cuckoo, to maneuvre into the nest. Perhaps it pushes the nest a little out of shape, but is still able to use the site for its own functions of stop signal decoding and for facilitating the release of the polypeptide. The term molecular mimicry has been dominant in the literature for a period of important advances in the understanding of protein synthesis. When the first structures of the ribosome appeared, the concept survived and was seen to be valid still. Now, we are at the stage of understanding the more detailed molecular interactions between ligands and the rRNA in particular, and how subtle changes in localized spatial orientations of atoms occur within these interactions. The simplicity of the original concept of mimicry will inevitably be blurred by this more detailed analysis. Nevertheless, it has provided a significant set of principles that allowed development of experimental programs to enhance our understanding of the dynamic events at this remarkable active site at the interface between the two subunits of this fascinating cell organelle, the ribosome.

In two Escherichia coli genomes, laboratory strain K-12 and pathological strain O157:H7, tandem termination codons as a group are slightly over-represented as termination signals. Individually however, they span the range of representations, over, as expected, or under, in one or both of the strains. In vivo, tandem termination codons do not make more efficient signals. The second codon can act as a backstop where readthrough of the first has occurred, but not at the expected efficiency. UGAUGA remains an enigma, highly over-represented, but with the second UGA a relatively inefficient back up stop codon.

The synthesis of release factor-2 (RF-2) in bacteria is regulated by a high efficiency +1 frameshifting event at an in-frame UGA stop codon. The stop codon does not specify the termination of synthesis efficiently because of several upstream stimulators for frameshifting. This study focusses on whether the particular context of the stop codon within the frameshift site of the Escherichia coli RF-2 mRNA contributes to the poor efficiency of termination. The context of UGA in this recoding site is rare at natural termination sites in E.coli genes. We have evaluated how the three nucleotides downstream from the stop codon (+4, +5 and +6 positions) in the native UGACUA sequence affect the competitiveness of the termination codon against the frameshifting event. Changing the C in the +4 position and, separately, the A in the +6 position significantly increase the termination signal strength at the frameshift site, whereas the nucleotide in the +5 position had little influence. The efficiency of particular termination signals as a function of the +4 or +6 nucleotides correlates with how often they occur at natural termination sites in E.coli; strong signals occur more frequently and weak signals are less common.

The translational stop signal and polypeptide release factor (RF) complexed with Escherichia coli ribosomes have been shown to be in close physical contact by site-directed photochemical cross-linking experiments. The RF has a protease-sensitive site in a highly conserved exposed loop that is proposed to interact with the peptidyltransferase center of the ribosome. Loss of peptidyl–tRNA hydrolysis activity and enhanced codon–ribosome binding by the cleaved RF is consistent with a model whereby the RF spans the decoding and peptidyltransferase centers of the ribosome with domains of the RF linked by conformational coupling. The cross-link between the stop signal and RF at the ribosomal decoding site is influenced by the base following the termination codon. This base determines the efficiency with which the stop signal is decoded by the RF in both mammalian and bacterial systems in vivo. The wide range of efficiencies correlates with the frequency with which the signals occur at natural termination sites, with rarely used weak signals often found at recoding sites and strong signals found in highly expressed genes. Stop signals are found at some recoding sites in viruses where −1 frame-shifting occurs, but the generally accepted mechanism of simultaneous slippage from the A and P sites does not explain their presence here. The HIV-1 gag-pol −1 frame shifting site has been used to show that stop signals significantly influence frame-shifting efficiency on prokaryotic ribosomes by a RF-mediated mechanism. These data can be explained by an E/P site simultaneous slippage mechanism whereby the stop codon actually enters the ribosomal A site and can influence the event.Key words: translational stop signal, decoding, release factor, frame-shifting.

Funding: This work was supported by University of St Andrews Restarting Research Funding Scheme (SARRF), funded through the SFC grant reference SFC/AN/08/020 (XRR064) and European Research Council grant ADOR (Advanced Grant 787073). The authors acknowledge the EPSRC Light Element Analysis Facility Grant (EP/T019298/1) and the EPSRC Strategic Equipment Resource Grant (EP/R023751/1). Since the outbreak of SARS-CoV-2, a multitude of strategies have been explored for the means of protection and shielding against virus particles: filtration equipment (PPE) has been widely used in daily life. In this work, we explore another approach in the form of deactivating coronavirus particles through selective binding onto the surface of metal–organic frameworks (MOFs) to further the fight against the transmission of respiratory viruses. MOFs are attractive materials in this regard, as their rich pore and surface chemistry can easily be modified on demand. The surfaces of three MOFs, UiO-66(Zr), UiO-66-NH2(Zr), and UiO-66-NO2(Zr), have been functionalized with repurposed antiviral agents, namely, folic acid, nystatin, and tenofovir, to enable specific interactions with the external spike protein of the SARS virus. Protein binding studies revealed that this surface modification significantly improved the binding affinity toward glycosylated and non-glycosylated proteins for all three MOFs. Additionally, the pores for the surface-functionalized MOFs can adsorb water, making them suitable for locally dehydrating microbial aerosols. Our findings highlight the immense potential of MOFs in deactivating respiratory coronaviruses to be better equipped to fight future pandemics. Publisher PDF

GDP-mannose-3',5'-epimerase (GME) from Arabidopsis thaliana catalyzes the epimerization of both the 3' and 5' positions of GDP-alpha-D-mannose to yield GDP-beta-L-galactose. Production of the C5' epimer of GDP-alpha-D-mannose, GDP-beta-L-gulose, has also been reported. The reaction occurs as part of vitamin C biosynthesis in plants. We have determined structures of complexes of GME with GDP-alpha-D-mannose, GDP-beta-L-galactose, and a mixture of GDP-beta-L-gulose with GDP-beta-L-4-keto-gulose to resolutions varying from 2.0 to 1.4 A. The enzyme has the classical extended short-chain dehydratase/reductase (SDR) fold. We have confirmed that GME establishes an equilibrium between two products, GDP-beta-L-galactose and GDP-beta-L-gulose. The reaction proceeds by C4' oxidation of GDP-alpha-D-mannose followed by epimerization of the C5' position to give GDP-beta-L-4-keto-gulose. This intermediate is either reduced to give GDP-beta-L-gulose or the C3' position is epimerized to give GDP-beta-L-4-keto-galactose, then C4' is reduced to GDP-beta-L-galactose. The combination of oxidation, epimerization, and reduction in a single active site is unusual. Structural analysis coupled to site-directed mutagenesis suggests C145 and K217 as the acid/base pair responsible for both epimerizations. On the basis of the structure of the GDP-beta-L-gulose/GDP-beta-L-4-keto-gulose co-complex, we predict that a ring flip occurs during the first epimerization and that a boat intermediate is likely for the second epimerization. Comparison of GME with other SDR enzymes known to abstract a protein alpha to the keto function of a carbohydrate identifies key common features.