Hepatocytes prepared by a simplified enzymatic technique were active in the incorporation of RNA and protein precursors into acid-insoluble material. The incorporation of RNA precursors was very markedly inhibited by low levels of aflatoxin B1 and G1 but not by aflatoxins B2 and G2. The activity of mixed function oxidases (MFO), the drug-metabolizing system of the endoplasmic reticulum, could be suppressed in these cells by SKF525A or stimulated by NADPH. SKF525A caused a reduction in the inhibition by aflatoxin B1 of the incorporation of RNA precursor into macromolecules. This finding suggests that a metabolite of aflatoxin B1 is the actual inhibitor of RNA synthesis in the cells. Measurement of lactate dehydrogenase activity showed these cells to be leaky on incubation at 37 degrees C and thus not suitable for studies of protein secretion.


This short essay examines infant formula marketing and information sources for their representation of "choice" in the infant feeding context, and finds that while providing information about breast and bottle feeding, infant formula manufacturers focus on mothers' feelings and intuition rather than knowledge in making decisions. In addition, the essay considers how "choice" operates in the history of reproductive rights, shifting the discourse from a rights-based set of arguments to one based on a consumerist mentality. Utilizing the work of historian Rickie Solinger and a 2007 paper for the National Bureau of Labor Statistics, I argue that the structure of market work, and not abstract maternal decision making, determine mothers' choices and practices concerning infant feeding. For true freedoms for mothers to be achieved, freedoms that would include greater social provisions for mothers, our culture will have to confront how structural constraints make breastfeeding difficult, as well as how the concept of choice divides mothers into those who make good choices and those who do not. I began to prepare this presentation with the idea that choice, as a concept, has been appropriated by infant formula companies in connection with women's liberation. Yet websites by Ross Laboratories, Mead Johnson, and Nestle/Carnation present "the choice to feed a baby with infant formula" as being increasingly under fire, presumably by strong medical evidence about the health contributions of breastfeeding. In this context, "choice" is presented as based on maternal feelings; mothers make good choices when they follow their sensibility, or "heart," rather than their heads.Specifically, current rhetoric from infant formula company websites articulates choice defensively, or with qualifiers about what is actually important in infant feeding. For example, in some articulations, love is more significant than breast milk or infant formula, which displaces the choice altogether onto the question of maternal emotions and, at some level, fitness. Implicitly, there's the sense of attack – if you feed with formula, you do not love your baby enough. The response, then, is to argue that it is love that counts in infant feeding and care, not what goes into the baby. Palpable in this rhetoric is the sense that mothers feel burdened by the need to demonstrate that they are good mothers by the fact that they love their children.Important in these materials is the notion of an "informed decision" to breastfeed or feed with infant formula. The information on each site about advantages and disadvantages of each choice exists to help mothers make these "informed decisions." However, the notion of an informed decision is undermined by the idea that decisions about infant feeding are made by the heart, not the head: in the end, information is not really the deciding factor. For example, on Mead Johnson's Enfamil website, after the discussion of "Choosing to Breastfeed," the page ends with the following statement: "Now that you're familiar with some of the advantages of breastfeeding, you have the added advantage of making an informed decision. In the end, of course, you'll do what your heart tells you. You can't go wrong. After all, that's the organ that's pumping out all that love for your baby" .Another Enfamil web page discusses "It's Your Family's Decision": "Only mom and dad know what will work best for their family. So, be confident in the choice that you make. The best way to deal with people who question your choice is to simply tell them, politely but firmly, that you have discussed how to feed your baby with your baby's doctor. Feel good about your decision and be confident your baby is getting the essential nutrients he needs" . ReadingRoe v. Wade made abortion legal in the USA. Rickie Solinger has shown that "choice" (as a concept) obscures the importance of rights with respect to abortion and reproductive freedom. In an article "Poisonous Choice," and her subsequent book Beggars and Choosers, Solinger criticizes the use of "choice" to articulate abortion rights, and demonstrates how "choice" operates to stratify mothers into categories of good and bad choosers (i.e. good and bad mothers) [Roe v. Wade. The use of "choice" instead of "abortion rights" made an alliance between the right to control one's fertility through pregnancy termination with the consumerist connotations of "choice." Any decision a woman makes about reproduction thus becomes vaguely connected to her "rights" as a consumer, rather than her rights as a human being [Thus "choice" concerning infant feeding in these product-oriented informational sites echoes some aspects of the discourses of reproductive rights struggles. Some feminist historians have struggled with the concept of choice with respect to abortion rights, seeing it as a figuration of consumerism right from the 1970s, when mothers) ,4. Firstan being .s. The rhetorical links of "choice" to consumerism, and the ability to turn the discourse of choice into one of blame led to "choice" becoming a very narrow political slogan that foregrounded the experience of middle-class women and made all other women vulnerable, because what are construed as "rights" in this scenario turn out to be privileges available to very few [Then Solinger argues that the articulation of abortion rights as "choice" opened up the possibility of women being criticized for making wrong choicevery few .rhetorical link made by infant formula companies between replacement feeding and maternal freedoms. The link exists materially in the structure of market work. In a recent "working paper" published in 2007 by the U.S. National Bureau of Labor Statistics, Albanesi and Olivetti demonstrate that life decisions and practices associated with increasing freedom for women – largely understood as participation in waged labor – were made possible by technological advances perceived to free women from domestic burdens, including the reproductive burden of breastfeeding [So what are the links between infant formula use and women's liberation, then? Is there no connection between infant feeding choice and women's freedom? It seems to me that there does not need to be a tfeeding . The restfeeding .Additionally, by identifying the development and marketing of infant formula as a necessary corollary to women's increased involvement in waged labor, Albanesi and Olivetti demonstrate how infant formula is linked to perceptions of women's liberation . In the What would real liberation for mothers entail? Structural changes to market work and greater social provisions, sponsored by the state, for mothers and their families, would lead to increased freedoms for mothers. However, even societies with generous maternity provisions continue to identify breastfeeding as a risky behavior, thus perpetuating a widespread distrust of nursing. This suggests that instituting breastfeeding as a norm will entail cultural changes in the way we think about women and their bodies – in public spaces, as nurturers, as sexual beings, as autonomous adults who are nevertheless physically connected to infants. Most perceived risks about women's bodies as breastfeeding mothers surround anxieties about modern maternity. Cultural change is needed, as feminism has always argued, to perceive women as responsible individuals fully capable of living their lives as free people. What we need to do is find new definitions of freedom that incorporate dependence, chance, and connectedness as essential and ordinary, so that women's rights as mothers are not abrogated by normative expectations of their fitness to carry out those roles.Significantly, choice is not liberation. If Rickie Solinger is correct, "choice" has only meant the right of access to abortion for middle class women, and thus it has never really meant choice, nor rights, in a universal sense . "ChoiceConstraints on breastfeeding are far harder to overcome for poor women, young women, women of color, and women with less education than for those women with education, resources, and more life experience. We can apply Solinger's analysis of abortion to infant feeding and notice that "choice" in infant feeding method operates to distinguish women who make "good choices" from those who do not, as if those choices are unconstrained. Infant feeding choices – whether made by "heart" or "head" – are practiced in the context of the social, cultural, and economic forces that structure most people's daily lives and intimate decisions. It is our responsibility, as feminists, to identify the constraints that reveal the "choice" itself to be not so much a choice but a class privilege, and then to figure out how to challenge the status quo that makes it so.The author declares that she has no competing interests.


The eukaryotic La protein recognizes the 3′ poly(U) sequences of nascent RNA polymerase III transcripts to assist folding and maturation. The 3′ ends of such RNAs are bound by the N-terminal domain of La (LaNTD). We have solved the crystal structures of four LaNTD:RNA complexes, each containing a different single-stranded RNA oligomer, and compared them to the structure of a previously published LaNTD:RNA complex containing partially duplex RNA. The presence of purely single-stranded RNA in the binding pocket at the interface between the La motif and RRM domains allows significantly closer contact with the 3′ end of the RNA. Comparison of the different LaNTD:RNA complexes identifies a conserved set of interactions with the last two nucleotides at the 3′ end of the RNA ligand that are key to binding. Strikingly, we also observe two alternative conformations of bound ssRNA, indicative of an unexpected degree of plasticity in the modes of RNA binding. The La protein is a highly abundant nuclear phosphoprotein that is conserved in eukaryotes. Originally identified as an autoantigen associated with rheumatic diseases such as lupus erythematosus and Sjögren's syndrome, La has been shown to have key roles in the processing and use of coding and noncoding RNAs . PerhapsIn yeast, La has also been shown to bind to the 3′ poly(U) ends that become exposed during endonucleolytic processing of pol II transcripts , again serving to stabilize the RNAs against exonucleolytic nibbling .internal RNA sequences within the untranslated regions of target mRNAs. Thus, La exhibits remarkable versatility in its modes of binding to RNA.In addition to its roles in the maturation of noncoding transcripts, La can directly facilitate translation of specific cellular  and viraHuman La (hLa) is a 408 amino acid monomeric modular protein containing three structured domains: a La motif and two RNA recognition motifs (RRMs 1 and 2); these are followed by a largely unstructured C-terminal tail that possesses a short basic motif (SBM) and a nuclear localization signal (NLS) . In receOH. Although largely consistent with previous structural and mutagenic analyses , it adopts an α-helical fold (residues 102–110) in the presence of short RNA oligomers   and the main-chain amide of I140 (RRM1), respectively, a pair of interactions that clearly helps to draw the two domains together. Notably, both these hydrogen bonds are significantly shorter in the presence of ssRNA ligands (∼2.8 Å) compared to the interactions observed when partial dsRNA is bound (∼3.1 Å)  with R57 from the La motif. Thus, the recognition of U−2 in the context of ssRNA engages a concerted set of protein:RNA and protein:protein interactions involving residues from both domains to form a tightly defined pocket that is specific in size, shape, and hydrogen-bonding capacity for a uridylate base  , illustrLa motif A. This lLa motif . The U−2ate base C. NMR anof LaNTD .−3 base to stack onto U−1 in an offset or half-staggered conformation. In the complexes formed with ssRNA, U−1 is also found to be stacked underneath an RNA base, but there are notable differences in the way that this is achieved by different RNA oligomers.In the previously published structure, the 3′ end of the RNA was found to make a tight turn to allow the U−4 rather than U−3 stacks on U−1. In this case, U−3 is flipped out and makes no specific contacts with the protein , this now allows U−4 to make a base-specific hydrogen bond with the backbone of the La motif , a tight turn in the RNA backbone was observed D, allowial dsRNA . HoweverA ligand D. In face duplex E.−3 is stacked on U−1. This finding suggests that the packing environment can have a considerable influence on the bound conformation. It also indicates that the interaction made by the stacked base (U−3 or U−4) may be relatively weak, a finding that is consistent with binding measurements (see below).The LaNTD:RNA complexes involving AUUUU and AUAAUUU occur in different space groups, C222 and C2, respectively , making 1212) further illustrated the potential impact of packing environment on the analysis of such complexes. In this case, although U−1 and U−2 were found to adopt the same conformations as observed in all other complexes  were used to obtain information about the dynamics of the La motif and RRM1 domains of LaNTD in the absence and presence of either UUU or UUUU RNA oligomers  ligands shows some small differences in the chemical shifts of the residues lining the binding cleft , suggestive of an alternative binding conformation (involving U−4/U−1 rather than C−3/U−1 stacking). The complex with UU also revealed a few minor differences in chemical shifts of the protein amide resonances in the binding pocket compared to longer oligo(U) ligands; this is likely to be because of the absence of uridylate residues preceding U−2 in this oligomer. Moreover, the spectrum obtained with AUUUU exhibits splitting of several resonances associated with residues in and around the binding site , suggestive of a slow conformational equilibrium between two states, possibly reflecting the two modes of binding observed in the crystal structures  . Althougructures .−1/U−3 or U−1/U−4 stacking interactions. The variation observed suggests that, although U−3 or U−4 may make base-specific contacts with the La motif (see above), these may not contribute much to binding affinity. We probed this structural interpretation by measuring the binding affinities of RNA decamers containing different sequences of the four nucleotides at their 3′ ends. Strikingly, although the binding affinity did indeed increase with the number of uridylates at the 3′ end (4 > 3 > 2), the benefit of adding a third or fourth uridylate was rather modest , which consistently make the closest contacts with the protein and appear to provide the majority of the binding energy for the interaction. Of these two nucleotides, only U−2 makes base-specific contacts with the protein; U−1 certainly contributes to binding but does so in a base-independent manner, by stacking on F35 and hydrogen bonding via backbone hydroxyl and phosphate groups to the La motif  requires at least four uridylates at the 3′ end for high-affinity binding  shows that the RNA generally lies across the β sheet surface in a 5′-3′ direction that would place the 3′ end of the RNA close to the site in the interdomain cleft that recognizes this feature . AlthougEscherichia coli strain BL21 (DE3) for 3 hr after induction with 1 mM IPTG .The N-terminal domain of human La protein  was amplified from pET-La(1–408) using PCR and subcloned into a modified pETM-11 vector which adds a thrombin-cleavable N-terminal 6× His tag . The pro2, 50 mM Tris (pH 7.5). This leaves a Gly-Ser dipeptide at the N terminus of the processed protein. The cleaved His tag was removed by reapplying the sample onto TALON resin; the LaNTD protein was then loaded onto a 10 ml HiTrap heparin column  in 20 mM Tris-HCl (pH 7), 100 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA and eluted with a 0.1–2 M KCl gradient over ten column volumes. The purified protein was buffer exchanged into 20 mM Tris-HCl (pH 7), 50 mM NaCl, 1 mM MgCl2, 1 mM dithiothreitol and concentrated to 12.5 mg/ml.Cell pellets were resuspended in 25 mM Tris-HCl (pH 7.6), 250 mM NaCl. Protease inhibitor cocktail (Sigma) and 0.01% Triton X-100 were added immediately prior to sonication. The protein was purified from the soluble fraction of the cell lysates using TALON metal affinity resin (BD Biosciences). The N-terminal His tag was removed by incubating in the presence of 10 U Thrombin (Sigma) per mg of protein during dialysis against 100 mM KCl, 2 mM CaClThe slightly longer form of LaNTD (La 1–194) used in NMR studies and gel binding assays was expressed and purified as described previously .3) grew within 2 weeks of streak seeding into similar drops with 2.6–2.8 M ammonium sulfate. LaNTD:UUUUUUUU complex crystals were grown in 0.1 M MMT buffer , 32% (w/v) PEG 1500 at 20°C. Typical crystal dimensions were 0.3 × 0.4 × 0.2 mm3. LaNTD:AUAAUUU and LaNTD:AUAUUUU complexes were crystallized in 0.2 M NaCl, 0.1 M phosphate citrate (pH 5.0), 22% (v/w) PEG 8000, 0.01 M taurine. Because the two oligomers differ only by one nucleotide (A or U at the 5′ end), crystals of the LaNTD:AUAUUUU complex were obtained by streak seeding from LaNTD:AUAAUUU crystals. In both cases, crystals grew as thin needles with typical dimensions 0.7 × 0.05 × 0.02 mm3.RNA oligomers  were synthesized, purified, and deprotected by Dharmacon Research. LaNTD:RNA complexes for crystallization were prepared at a molar ratio of 1:1. All crystallizations were performed using sitting-drop vapor diffusion. Crystals of the LaNTD:AUUUU complex grew spontaneously in 2.7 M ammonium sulfate, 0.1 M sodium citrate (pH 6.0) at 4°C; larger crystals  glycerol. X-ray diffraction data were collected at the European Synchrotron Radiation Facility, Grenoble, France.Diffraction data were processed using the CCP4 package . In ever1, T2, and {1H}-15N NOE) were acquired on a Bruker Avance at 14.1 T using the pulse sequences adapted from standard schemes (2) were measured both for apo- and RNA-bound protein at 0.6, 0.4, and 0.2 mM. Because the rates at the two lower protein concentrations were very similar (with and without RNA), all measurements were performed at 0.2 mM. R2 were measured with eight different total CPMG relaxation delays ranging from 6.36 to 82.68 ms. The longitudinal relaxation rates (R1) were measured with relaxation delays ranging from 10 to 1100 ms, typically in eight evenly spaced points. In both experiments, the shortest and the longest points were repeated for error calculations. Relaxation rates were calculated from peak intensity decay curves fit using CurveFit . Uncertainties were calculated using jackknife simulations  (Relaxation experiments (T schemes . To deteulations . HYDRONMulations  predictictively) ; the preRNA oligomers were synthesized and gel purified by Dharmacon. The assays were performed using protocols reported previously . Each exϕ is the fraction of bound RNA determined by the intensity of the protein-bound RNA signal divided by the total RNA signal (both bound and free), P is the protein concentration, and N is the RNA concentration. Theoretical fitting was performed using least-squares analysis. The RNA concentration was estimated to be 65% of the calculated concentration  and then fitted iteratively using least-squares analysis. Relative dissociation constants were calculated using CACACAAUUU as a reference point, assuming that this oligomer bound LaNTD in a single conformation. Typical RNA concentrations were around 1 nM.Dissociation constants were determined by plotting the percent of bound RNA versus the protein concentrations. The curves were fit using the following equation :ϕ=(P+N+K


For some viruses this effect has been attributed to interference with viral polyprotein processing. In this study we demonstrate that the combination of Zn2+ and PT at low concentrations (2 µM Zn2+ and 2 µM PT) inhibits the replication of SARS-coronavirus (SARS-CoV) and equine arteritis virus (EAV) in cell culture. The RNA synthesis of these two distantly related nidoviruses is catalyzed by an RNA-dependent RNA polymerase (RdRp), which is the core enzyme of their multiprotein replication and transcription complex (RTC). Using an activity assay for RTCs isolated from cells infected with SARS-CoV or EAV—thus eliminating the need for PT to transport Zn2+ across the plasma membrane—we show that Zn2+ efficiently inhibits the RNA-synthesizing activity of the RTCs of both viruses. Enzymatic studies using recombinant RdRps (SARS-CoV nsp12 and EAV nsp9) purified from E. coli subsequently revealed that Zn2+ directly inhibited the in vitro activity of both nidovirus polymerases. More specifically, Zn2+ was found to block the initiation step of EAV RNA synthesis, whereas in the case of the SARS-CoV RdRp elongation was inhibited and template binding reduced. By chelating Zn2+ with MgEDTA, the inhibitory effect of the divalent cation could be reversed, which provides a novel experimental tool for in vitro studies of the molecular details of nidovirus replication and transcription.Increasing the intracellular Zn 2+ concentrations are known to efficiently impair replication of a number of RNA viruses, e.g. by interfering with correct proteolytic processing of viral polyproteins. Here, we not only show that corona- and arterivirus replication can be inhibited by increased Zn2+ levels, but also use both isolated replication complexes and purified recombinant RdRps to demonstrate that this effect may be based on direct inhibition of nidovirus RdRps. The combination of protocols described here will be valuable for future studies into the function of nidoviral enzyme complexes.Positive-stranded RNA (+RNA) viruses include many important pathogens. They have evolved a variety of replication strategies, but are unified in the fact that an RNA-dependent RNA polymerase (RdRp) functions as the core enzyme of their RNA-synthesizing machinery. The RdRp is commonly embedded in a membrane-associated replication complex that is assembled from viral RNA, and viral and host proteins. Given their crucial function in the viral replicative cycle, RdRps are key targets for antiviral research. Increased intracellular Zn Nevertheless, the intracellular concentration of free Zn2+ is maintained at a relatively low level by metallothioneins, likely due to the fact that Zn2+ can serve as intracellular second messenger and may trigger apoptosis or a decrease in protein synthesis at elevated concentrations 2+ concentrations and the addition of compounds that stimulate cellular import of Zn2+, such as hinokitol (HK), pyrrolidine dithiocarbamate (PDTC) and pyrithione (PT), were found to inhibit the replication of various RNA viruses, including influenza virus 2+ levels affect a common step in the replicative cycle of these viruses.Zinc ions are involved in many different cellular processes and have proven crucial for the proper folding and activity of various cellular enzymes and transcription factors. Zn2+ uptake within minutes and inhibits RNA virus replication through a mechanism that has only been studied in reasonable detail for picornaviruses In vitro studies with purified rhinovirus and poliovirus 3C proteases revealed that protease activity was inhibited by Zn2+2+ import was therefore hypothesized to result from inhibition of the viral RNA-dependent RNA polymerase (RdRp) and cellular cofactors 2+ on the activity of purified RdRps from rhinoviruses and hepatitis C virus was noted, but not investigated in any detail In cell culture, PT stimulates ZnDetails on the effect of zinc ions are currently largely unknown for nidoviruses. This large group of positive-strand RNA (+RNA) viruses includes major pathogens of humans and livestock, such as severe acute respiratory syndrome coronavirus (SARS-CoV), other human coronaviruses, the arteriviruses equine arteritis virus (EAV), and porcine reproductive and respiratory syndrome virus (PRRSV) 2+ is a potent inhibitor of the replication of SARS-coronavirus (SARS-CoV) and equine arteritis virus (EAV) in cell culture. To assess whether - besides a possible effect on proteolytic processing - nidovirus RTC subunits and RNA synthesis are directly affected by Zn2+, we employed in vitro systems for SARS-CoV and EAV RNA synthesis that are based on membrane-associated RTCs isolated from infected cells (from here on referred to as RTC assays) in vitro recombinant RdRp assays to directly study the effect of zinc ions on the RdRps of SARS-CoV and EAV Analogous to picornaviruses in vitro approaches, we were able to demonstrate that Zn2+ directly impairs nidovirus RNA synthesis, since it had a strong inhibitory effect in both RTC and RdRp assays. Interestingly, the Zn2+-mediated inhibition could be reversed through the addition of a Zn2+ chelator (MgEDTA). We therefore applied this compound to stop and restart the in vitro RNA-synthesizing activity at will. This convenient tool allowed us to study various mechanistic aspects of arteri- and coronavirus RNA synthesis in more detail. Additionally, the zinc-mediated inhibition of nidovirus RNA synthesis described here may provide an interesting basis to further explore the use of zinc-ionophores in antiviral therapy.Using these independent 2+ is maintained at a relatively low level by metallothioneins 2+ and compounds that stimulate import of Zn2+ into cells, such as PT, were previously found to inhibit replication of several picornaviruses, including rhinoviruses, foot-and-mouth disease virus, coxsackievirus, and mengovirus in cell culture 2+ has a similar effect on nidoviruses, we investigated the effect of PT and Zn2+ on the replication of EAV and SARS-CoV in Vero-E6 cells, using reporter viruses that express green fluorescent proteins (GFP), i.e., EAV-GFP Zinc ions are involved in many different cellular processes, but the concentration of free Zn2. Treatment with PT of concentrations up to 32 µM in combination with <4 µM ZnOAc2 did not reduce the viability of mock-infected cells after 18 h .We first assessed the cytotoxicity of a range of PT concentrations (0–32 µM) in the presence of 0 to 8 µM ZnOActer 18 h , as meas2 on EAV-GFP and SARS-CoV-GFP replication. To this end, Vero-E6 cells in 96-well plates were infected with a multiplicity of infection (m.o.i.) of 4. One hour post infection (h p.i.), between 0 and 32 µM of PT and 0, 1, or 2 µM ZnOAc2 were added to the culture medium. At 17 h p.i., a time point at which GFP expression in untreated infected cells reaches its maximum for both viruses, cells were fixed, and GFP fluorescence was quantified.Using these non-cytotoxic conditions we subsequently tested the effect of PT and ZnOAc2+ was added to the medium. We found that addition of ZnOAc2 alone also reduced virus replication, but only at levels that were close to the 50% cytotoxicity concentration (CC50) of ZnOAc2 in Vero-E6 cells . This is likely due to the poor solubility of Zn2+ in phosphate-containing medium and the inefficient uptake of Zn2+ by cells in the absence of zinc-ionophores. The combination of 2 µM PT and 2 µM ZnOAc2 resulted in a 98±1% and 85±3% reduction of the GFP signal for EAV-GFP and SARS-CoV-GFP, respectively. No cytotoxicity was observed for this combination of PT and ZnOAc2 concentrations. From the dose-response curves in 50 value of 82 µM was calculated for PT in the presence of 2 µM zinc. Half maximal inhibitory concentrations (IC50) of 1.4 µM and 0.5 µM and selectivity indices of 59 and 164 were calculated for SARS-CoV and EAV, respectively.The reporter gene expression of both SARS-CoV-GFP and EAV-GFP was already significantly inhibited in a dose-dependent manner by the addition of PT alone . This efin vitro RNA-synthesizing activity of RTCs isolated from cells infected with SARS-CoV or EAV 32P]CMP is incorporated into both genomic (replication) and sg mRNA (transcription)  . This alboth EAV  and SARSboth EAV , a dose-2+ was reversible, RTC reactions were started in the presence or absence of 500 µM Zn2+. After 30 min, these reactions were split into two aliquots and magnesium-saturated EDTA (MgEDTA) was added to one of the tubes to a final concentration of 1 mM , most likely by chelating the Mg2+ that is crucial for RdRp activity 2+  of SARS-CoV-infected cells, as this increase was not observed when MgEDTA was added to a control reaction without additional Zn2+  and EAV (nsp9) using previously developed RdRp assays 32P]AMP into RNA products of up to 18 nt in length , after which the reaction was split in two. Then, [α-32P]CTP was added to both tubes (pulse), 500 µM Zn2+ was added to one of the tubes, and samples were taken at different time points during the reaction  produced in the presence of Zn2+, since multiple initiation events are required on these short molecules to obtain signal intensities similar to those resulting from a single initiation event on the long genomic RNA, e.g., 16 times more in the case of RNA7. In contrast to EAV, the effect of Zn2+ on RNA synthesis by SARS-CoV RTCs was not limited to initiation, but appeared to impair the elongation phase as well, given that the addition of Zn2+ completely blocked further incorporation of [α-32P]CMP when added 40 min after the start of the reaction  SARS-CoV nsp12 RdRp activity was restricted to the addition of only a single nucleotide to the primer de novo initiating RdRps 2+ on initiation and elongation by performing an experiment in which a pulse with a low concentration of [α-32P]ATP was followed by a chase in the presence of 50 µM of unlabeled ATP, which increased processivity and allowed us to study elongation  in the presence and absence of Zn2+ .To assess whether Zn of Zn2+ . To meashe assay . We also6-tagged nsp9, and radiolabeled EAV genomic RNA or various short RNA templates including polyU, whereas we were able to detect binding of a control protein  was previously observed for several picornaviruses such as rhinoviruses, foot-and-mouth disease virus, coxsackievirus, and mengovirus Zinc ions and zinc-ionophores, such as PT and PDTC, have previously been described as potent inhibitors of various RNA viruses. We therefore investigated whether PT-stimulated import of zinc ions into cells also inhibited the replication of nidoviruses in cell culture. Using GFP-expressing EAV and SARS-CoV toxicity . Inhibit2+ on the replication of picornaviruses appeared to be due to interference with viral polyprotein processing. In infections with the coronavirus mouse hepatitis virus (MHV), Zn2+ also interfered with some of the replicase polyproteins cleavages 2+) than used in our studies. Since impaired replicase processing will indirectly affect viral RNA synthesis in the infected cell, we used two recently developed in vitro assays to investigate whether Zn2+ also affects nidovirus RNA synthesis directly. Our in vitro studies revealed a strong inhibitory effect of zinc ions on the RNA-synthesizing activity of isolated EAV and SARS-CoV RTCs. Assays with recombinant enzymes subsequently demonstrated that this was likely due to direct inhibition of RdRp function. The inhibitory effect could be reversed by chelating the zinc ions, which provides an interesting experimental (on/off) approach to study nidovirus RNA synthesis. Addition of Zn2+ following initiation of EAV RNA synthesis had little or no effect on NTP incorporation in molecules whose synthesis had already been initiated in the absence of Zn2+ , as was also reported for the poliovirus RdRp 2+ was even observed at low concentrations and in the presence of a more than 25-fold excess of Mg2+, suggesting that either the affinity of the active site for Zn2+ is much higher or that Zn2+ does not compete for Mg2+-binding and binds to another zinc(-specific) binding site in the protein.It has been well established that DNA and RNA polymerases use a triad of conserved aspartate residues in motifs A and C to bind divalent metal ions like Mg2+ coordinating residues in the Zn2 zinc-binding pocket found in motif E of the Dengue virus RdRp viral RNA synthesis, and revealed novel mechanistic differences between the RdRps of SARS-CoV and EAV.In summary, the combination of zinc ions and the zinc-ionophore PT efficiently inhibits nidovirus replication in cell culture. This provides an interesting basis for further studies into the use of zinc-ionophores as antiviral compounds, although systemic effects have to be considered Vero-E6 cells were cultured and infected with SARS-CoV  or SARS-CoV-GFP as described previously 2. Infected cells were fixed at 17 h p.i. by aspirating the medium and adding 3% paraformaldehyde in PBS. After washing with PBS, GFP expression was quantified by measuring fluorescence with a LB940 Mithras plate reader (Berthold) at 485 nm. To determine toxicity of ZnOAc2 and PT, cells were exposed to 0–32 µM PT and 0–8 µM ZnOAc2. After 18 h incubation, cell viability was determined with the Cell Titer 96 AQ MTS assay (Promega). EC50 and CC50 values were calculated with Graphpad Prism 5 using the nonlinear regression model.One day prior to infection, Vero-E6 cells were seeded in transparent or black (low fluorescence) 96-well clusters at 10,000 cells per well. The next day, cells were infected with SARS-CoV-GFP or EAV-GFP with an m.o.i. of 4, and 1 h p.i. the inoculum was removed and 100 µl of medium containing 2% fetal calf serum (FCS) was added to each well. In some experiments 0–32 µM of pyrithione (Sigma) was added in addition to 0–2 µM ZnOAc5′-GCUAUGUGAGAUUAAGUUAU-3′), SAV481R (5′-UUUUUUUUUUAUAACUUAAUCUCACAUAGC-3′) and poly(U)18 (5′-UUUUUUUUUUUUUUUUUU-3′) were purchased from Eurogentec, purified from 7 M Urea/15% PAGE gels and desalted through NAP-10 columns . To anneal the RNA duplex SAV557R/SAV481R, oligonucleotides were mixed at equimolar ratios in annealing buffer , denatured by heating to 90°C and allowed to slowly cool to room temperature after which they were purified from 15% non-denaturing PAGE gels.RNA oligonucleotides SAV557R  was added to a final concentration of 1 mM. After RNA isolation, the 32P-labeled reaction products were separated on denaturing 1% (SARS-CoV) or 1.5% (EAV) agarose formaldehyde gels. The incorporation of [α-32P]CMP into viral RNA was quantified by phosphorimaging of the dried gels using a Typhoon scanner  and the ImageQuant TL 7 software .SARS-CoV and EAV RTCs were isolated from infected cells and assayed for activity E. coli BL21(DE3) with plasmid pDEST14-nsp9-CH was grown in auto-induction medium ZYM-5052 GCCCTGGAGAGTTGTGAT-3′5′-TACTGCCTTGAAACA and GGCTGTTTCAAGGCAGTA-3′5′-ATCACAACTCTCCAG, and plasmid pASK3-Ub-nsp12-CHis6 with oligonucleotides GCTTATCCAAAATGTG-3′5′-CCTTATGGGTTGG and AGCCCAACCCATAAGGA-3′5′-CACATTTTGGATA, as described elsewhere SARS-CoV nsp12 and EAV nsp9 were purified essentially as described elsewhere 2+ in this assay, 0.5 µl of a dilution series of 0–80 mM ZnOAc2 was added to the 5 µl reaction mixture, yielding final Zn2+ concentrations of 0–8 mM. The EAV RdRp assay contained 1 µM nsp9, 1 µM RNA template poly(U)18, 0.17 µM [α-32P]ATP , 50 µM ATP, 20 mM Tris-HCl (pH 8.0), 10 mM NaCl, 10 mM KCl, 1 mM MnCl2, 4 mM MgOAc2, 5% glycerol, 0.1% Triton-X100, 1 mM DTT and 0.5 units RNaseOUT. ZnOAc2 was added to the reaction to give a final concentration of 0–6 mM. To chelate Zn2+ during reactions, MgEDTA was added to a final concentration of 8 mM. Reactions were terminated after 1 hour and analyzed as described Standard reaction conditions for the RdRp assay with 0.1 µM of purified SARS-CoV nsp12 are described elsewhere 32P-labeled SAV557R/SAV481R RNA duplex, for 10 minutes at 30°C either in presence or absence of 6 mM ZnOAc2. Reactions were analyzed as described previously SARS-CoV RdRp was incubated with 0.2 nM 5′ Figure S1Effect of various divalent cations on the RdRp activity of SARS-CoV nsp12. Purified recombinant SARS-CoV nsp12 was incubated with a primed template, ATP, and [α-32P]ATP in the presence of either 6 mM Mg2+ only (lane 1), and with increasing concentrations of a second divalent metal (M2+), specifically: 2–6 mM Ca2+ (lane 2–4), 2–6 mM Co2+ (lane 5–7), 2–6 mM Zn2+ (lane 8–10), or 2–6 mM Mn2+ (lane 11–13). The strongest inhibition was observed for Zn2+. For more details on the SARS-CoV nsp12 RdRp assay, see the main text.(1.55 MB TIF)Click here for additional data file.Figure S22+ on the dinucleotide extension activity of EAV nsp9.Effect of Zn Purified recombinant EAV nsp9 was incubated with a U18 template in the presence of [α-32P]ATP, ATP, 4 mM Mg2+, 1 mM Mn2+, and 1 µM ApA. (A) Reaction mixtures were split into two aliquots, one of which was supplemented with 6 mM Zn2+, and samples were taken at the time points (minutes) indicated above the lanes. In the absence of Zn2+, EAV nsp9 initiates de novo and produces di- and trinucleotides, indicated with A2 and A3, respectively. A non-specific band, unrelated to RdRp activity, between A2 and A3 is indicated with an asterisk. In the presence of 6 mM Zn2+, the synthesis of dinucleotides and trinucleotides was blocked. (B) When performing the assay described under (A) in the absence of Zn2+, a full-length product of 18 nucleotides is formed. This product is not observed when the assay is performed in the presence of 6 mM Zn2+, but nsp9 was capable of elongating the provided dinucleotide primer ApA into tri- (ApA*pA) and tetranucleotide ((ApA*pA*pA) products (the asterisk indicates radiolabeled phosphates). Due to the absence of a 5′ triphosphate group, these reaction products migrate much slower in the 20% acrylamide and 7 M urea gel used for this analysis. See the main text for additional experimental details on the EAV nsp9 RdRp assay.(2.16 MB TIF)Click here for additional data file.Figure S3Putative zinc-binding residues in the predicted structure of SARS-CoV nsp12 and comparison with the structure of the zinc-containing Dengue virus RdRp domain. (A) Sequence alignment of coronavirus RdRps showing conservation of four potential zinc-binding residues amino acids  in the C-terminal region of coronavirus nsp12. Black shading indicates complete conservation among coronaviruses. The coronavirus RdRp sequences were aligned with Muscle 3.6. The aligned sequences and NCBI accession numbers are the following: mouse hepatitis virus strain A59 , human CoV 229E , infectious bronchitis virus strain Beaudette , bovine coronavirus , feline coronavirus , and SARS-CoV strain Frankfurt-1 . (B) Crystal structure of the Dengue virus RdRp domain showing the position of four cysteine and histidine residues that form Zn2+-binding pocket Zn2, located close to motif E (depicted in red). A second Zn2+-binding pocket (Zn1) and the two zinc ions identified in the crystal structure are indicated in blue-gray. (C) Predicted three-dimensional structure model of SARS-CoV nsp12 , based on PDB code 1O5S, rendered with Swiss-PdbViewer 4.01 and POV-Ray 3.6. The positions of the conserved cysteine and histidine residues indicated in panel A (C799-H810-C813-H816) close to motif E (depicted in red) and RdRp active-site residues  are indicated. The spatial arrangement of these cysteines and histidines in this model strikingly resembles the positioning of the metal ion-coordinating residues of Zn-binding pocket Zn2 in the Dengue virus RdRp domain (see panel B).(0.86 MB TIF)Click here for additional data file.


TOR1A typically manifests as early-onset generalized dystonia, DYT1 dystonia is genetically and clinically heterogeneous. Recently, another Exon 5 mutation (c.863G>A) has been associated with early-onset generalized dystonia and some ΔGAG mutation carriers present with late-onset focal dystonia. The aim of this study was to identify TOR1A Exon 5 mutations in a large cohort of subjects with mainly non-generalized primary dystonia.Although the c.904_906delGAG mutation in Exon 5 of TOR1A Exon 5 coding sequence in 1014 subjects with primary dystonia  and 250 controls . Diagnostic sensitivity and specificity were evaluated in an additional 8 subjects with known ΔGAG DYT1 dystonia and 88 subjects with ΔGAG-negative dystonia.High resolution melting (HRM) was used to examine the entire TOR1A Exon 5 showed high (100%) diagnostic sensitivity and specificity. HRM was rapid and economical. HRM reliably differentiated the TOR1A ΔGAG and c.863G>A mutations. Melting curves were normal in 250/250 controls and 1012/1014 subjects with primary dystonia. The two subjects with shifted melting curves were found to harbor the classic ΔGAG deletion: 1) a non-Jewish Caucasian female with childhood-onset multifocal dystonia and 2) an Ashkenazi Jewish female with adolescent-onset spasmodic dysphonia.HRM of TOR1A are rarely associated with non-generalized primary dystonia.First, HRM is an inexpensive, diagnostically sensitive and specific, high-throughput method for mutation discovery. Second, Exon 5 mutations in  Dystonia is a syndrome of sustained muscle contractions, frequently causing twisting and repetitive movements, or abnormal postures . DystoniIn most neurology subspecialty practices, the vast majority of patients with dystonia are adults with primary focal or segmental disease. Genetic factors likely play a major role in late-onset primary dystonia since 8–27% of patients with primary late-onset dystonia have one or more family members affected with dystonia -9 and seTOR1A is characteristically associated with early-onset generalized dystonia [TOR1A (c.863G>A) has been described in a female patient with severe childhood-onset generalized dystonia [TOR1A. Leung and colleagues [SGCE mutation [The ΔGAG (c.904_906delGAG) mutation in Exon 5 of dystonia . Althougdystonia -19. Anotdystonia . The G>Alleagues  reportedmutation . As descmutation .TOR1A gene in non-generalized primary dystonia, we used high resolution melting (HRM) to examine the entire TOR1A Exon 5 coding sequence in a large cohort of patients with non-generalized dystonia. HRM has been shown to be a fast and accurate, closed-tube, post-PCR mutation scanning technique that monitors the progressive change in fluorescence caused by the release of an intercalating DNA dye from DNA duplexes as they are denatured by minor increases in temperature [TOR1A are rare in non-generalized primary dystonia.To help address the role of the perature . Our resAll human studies were performed in accordance with institutional review board guidelines and all subjects gave informed consent. Subjects with dystonia and neurologically-normal controls were acquired from outpatient clinics at participating institutions  and support group meetings of the National Spasmodic Dysphonia Association (NSDA), National Spasmodic Torticollis Association (NSTA), Benign Essential Blepharospasm Research Foundation (BEBRF), Dystonia/Spasmodic Torticollis, and Dystonia Medical Research Foundation (DMRF). All subjects with dystonia acquired at support group meetings were examined by M.S.L. Subjects with Parkinson's disease and other movement disorders were acquired from the clinics of M.S.L. and R.F.P. Clinical diagnoses and classifications were made by means of history and examination by one or more neurologists and/or neurolaryngologist at each institution. Neurologically-normal control subjects were defined as individuals with no personal or first-degree family history of neurological disease. In addition, all control subjects acquired at the University of Tennessee Health Science Center and support group meetings were examined by M.S.L. or R.F.P. Dystonia was classified in accordance with established schemes ,3,24. SuClinical diagnoses for 1264 subjects interrogated with HRM appear in Table ® dsDNA Assay Kit  and agarose gel electrophoresis. High-quality DNA samples were diluted with PCR water to a concentration of 10 ng/μl. Poor quality samples were rescued by whole genome amplification with the REPLI-g® Mini Kit from Qiagen . Samples that could not be rescued were not used for HRM and do not appear in Table DNA was extracted from peripheral blood leucocytes using Roche's DNA Isolation Kit for Mammalian Blood . DNA quantity and quality were analyzed with a NanoDrop ND-100 spectrophotometer , Quant-iT™ PicoGreenTOR1A Exon 5 coding sequence . Another forward primer was synthesized for site-directed mutagenesis of Exon 5 to generate the c.863G>A mutation (aacagcaccttgtttcttcttcccaggtggcttctggcacagcagcttaattgaccggaacctcattgattattttgttcccttcctccccctggaatacaaacacctaaaaatgtgtatccgagtggaaatgcagtcccAaggctatga). To evaluate HRM results with amplicons of different sizes, a pair of previously-published primers was used to amplify the DYT1 ΔGAG region of Exon 5 [Using Primer3 (frodo.wi.mit.edu), a pair of PCR primers was designed to cover the entire f Exon 5 .® 480 Real-Time PCR system and High Resolution Master Mix from Roche Applied Science  were used for HRM. The master mix contains the LightCycler 480 ResoLight Dye. The High Resolution Melting Master Mix is a ready-to-use hot-start mix designed for PCR amplification followed by HRM curve analysis for detection of sequence variants. ResoLight, a novel saturating DNA dye, was specifically designed for detection of sequence variations by differences in melting curves. ResoLight can be used at high concentrations without inhibiting PCR and its homogeneous staining of target sequences results in sharp melting signals. As the temperature of the solution is increased during HRM, the specific sequence of the amplicon (primarily GC content and length) determines the melting behavior. When the fluorescence signal is plotted against temperature, fluorescence intensity decreases as double-stranded DNA (dsDNA) becomes single stranded and ResoLight is released. Since melting point (TM) differences are often too small for reliable differentiation of amplicons, separation is achieved by signal normalization and temperature shifting  using 10–20 ng of template DNA, 1× HRM Master Mix, 2.5 mM MgCl2 and 200 nM of each primer in a 20-μl reaction volume. PCR cycling and HRM conditions were carried out as follows: 95°C for 10 min; 45 cycles at 95°C for 10 s, 60°C for 15 s, and 72°C for 15 s; 95°C for 1 min, 40°C for 1 min, and the final HRM temperature ramp from 70°C to 95°C rising at 0.1°C/s. All subject samples and negative controls were run in duplicate using 96-well plates.The HRM Master Mix contains FastStart Taq DNA polymerase, reaction buffer, a dNTP mix and ResoLight. HRM was optimized to detect and differentiate the ΔGAG and c.863G>A mutations by adjusting reaction conditions and the concentrations of template DNA, primers and MgClWith LightCycler 480 Gene Scanning Software, melting curves and difference plots were analyzed by three investigators  blinded to phenotype. All samples were unambiguously assigned to one of three genotypes  by Gene Scanning Software. DNA from wells with abnormal melting curves was purified with the Qiagen QIAquick PCR Purification Kit. The ΔGAG deletion and c.863G>A mutations were confirmed by direct sequencing of purified PCR products in the forward and reverse directions using an ABI Prism 377 DNA Sequencer .As seen in Fig TOR1A Exon 5 amplicon  than the smaller amplicon  which was generated with previously published primers [HRM robustly distinguished samples with either ΔGAG deletions or c.863G>A mutations from normals Fig . Diagnos primers .® 480 HRM Master Mix  was purchased for $400  and contained reagents for 500 reactions . LightCycler® 480 Multiwell Plates 96, white with sealing foils , must be used for HRM and were purchased for $360/50 plates ($7.20/plate). Excluding the costs of pipette tips and primers, 96 reactions were completed for $84.00. On average, each plate was setup, run, and analyzed in 3 hrs.LightCyclerTOR1A. No TOR1A Exon 5 mutations or variants were identified in the remaining 1012 subjects with dystonia or 250 controls. Subject #1 with the classic DYT1 ΔGAG mutation exhibited multifocal dystonia. She was a 48-yr-old non-Jewish Caucasian female with cervical dystonia, spasmodic dysphonia (adductor subtype), and writer's cramp. Cervical dystonia became manifest at 12 yrs of age whereas laryngeal involvement and writer's cramp became apparent at 39 and 45 yrs of age, respectively. She reported consistent benefit from injections of botulinum toxin type A for treatment of her laryngeal and cervical dystonia. Her mother had never been diagnosed with "dystonia" but reportedly exhibited action-induced "leg tremors." A maternal aunt (her mother's monozygotic twin) suffered from an undiagnosed voice disorder prior to her death. Subject #1 had two siblings, both brothers. By report, one brother was neurologically normal. The second brother developed generalized dystonia at 9 years of age and died at 16 years of age with respiratory complications.Two subjects (#1 and #2) with non-generalized primary dystonia were found to harbor the classic DYT1 ΔGAG deletion in Exon 5 of Subject #2, also with the classic DYT1 ΔGAG mutation, was a 63-yr-old woman of Ashkenazi Jewish descent. She had spasmodic dysphonia (adductor subtype) that became manifest at 19 years of age. Injections of botulinum toxin type A achieved consistently excellent results over a period of 12 yrs. Subject #2 also exhibited a bilateral upper extremity action tremor (<2 cm excursions). She had one child who was neurologically normal. Her mother and maternal grandmother had received diagnoses of "essential tremor." None of her family members were available for neurological examination.de novo ΔGAG mutations of TOR1A Exon 5 have been described [TOR1A Exon 5 variants, including the recently described c.863G>A missense mutation, must also be rare in subjects with non-generalized primary dystonia.In 2000, Bressman and colleagues published diagnostic testing guidelines for the DYT1 GAG deletion  and recoescribed ,27, our As outlined in Table TOR1A Exon 5 mutations. Since the carrier frequency of the classic DYT1 ΔGAG mutation is estimated at 1:1000–1:3000 in Ashkenazi Jews [In comparison with several of the studies listed in Table azi Jews  and lessazi Jews , inclusiTOR1A and does not exclude a role for TOR1A mutations and/or variants in the etiopathogenesis of late-onset primary dystonia. Single nucleotide polymorphisms (SNPs) within or in close proximity to the 3' UTR of TOR1A may be associated with primary focal dystonia [TOR1A copy number variants and single nucleotide polymorphisms must be interrogated in future studies.Our study was limited to interrogation of Exon 5 of dystonia -36. MutaTOR1A mutations [Previous studies of primary dystonia have employed a wide variety of methods for detection of utations ,21,37-49utations -48 and Sutations ,21, are HRM has recently been introduced as a screening method for mutation detection. HRM is a closed-tube method that can be performed in a rapid, economical, reliable, and high-throughput fashion. In a comparative study, Chou and colleagues showed that HRM is superior to DHPLC . HRM hasTOR1A Exon 5 and generated a 314 bp amplicon. Theoretically, differentiation of samples carrying a sequence variation is better with shorter amplicons. In general, amplicons from 50 to 250 bp are ideal for HRM, particularly when employed for SNP and homozygous mutation analysis. However, 100% sensitivity and specificity has been observed with amplicons larger than 600 bp [The utility and efficiency of HRM can be maximized by careful experimental design. In our study, a single pair of primers was designed to cover the entire coding region of n 600 bp . Thus, t® 480 Real-Time PCR system offers 384-well plate capabilities which, when coupled with a robotic workstation, permits completion of very large projects  in a single week. Clearly, HRM is well-suited for many candidate gene and SNP-association studies of dystonia and other neurological disorders.HRM offers additional advantages over older screening techniques. First, amplicons of interest only require purification prior to follow-up sequencing. There is no need to PCR amplify DNA templates. Second, HRM robustly differentiates genotypes despite moderate differences in well-to-well concentrations of DNA templates. Lastly, Roche's LightCyclerTOR1A Exon 5 using HRM. HRM showed 100% diagnostic sensitivity and specificity. In a large cohort of patients with non-generalized primary dystonia in the US, TOR1A Exon 5 mutations were very uncommon; only two classic DYT1 ΔGAG and no c.863G>A mutations were identified. HRM may be applicable to high-throughput mutation detection in other movement and general neurological disorders.In conclusion, we have developed a fast, efficient and reliable screening method for known mutations (ΔGAG and c.863G>A) within the coding region of The authors declare that they have no competing interests.MSL designed the study. JX, SG and YZ extracted DNA from blood samples. JX and MSL optimized and implemented the HRM protocol and analyzed HRM data. RWB, JSP, BAR, SDT, MK, RP, AB, ZKW, RJU, PH, DKS, DT, DDT, KPF, RFP and MSL examined research subjects. SDB provided DNA from subjects with a clinical diagnosis of dystonia. JX and MSL drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Self-rated health (SRH) has been demonstrated to be an accurate reflection of a person's health and a valid predictor of incident mortality and chronic morbidity. We aimed to evaluate the distribution and factors associated with SRH and its association with biomarkers of cardio-metabolic diseases among middle-aged and elderly Chinese.Survey of 1,458 men and 1,831 women aged 50 to 70 years, conducted in one urban and two rural areas of Beijing and Shanghai in 2005. SRH status was measured and categorized as good (very good and good) vs. not good . Determinants of SRH and associations with biomarkers of cardio-metabolic diseases were evaluated using logistic regression.Thirty two percent of participants reported good SRH. Males and rural residents tended to report good SRH. After adjusting for potential confounders, residence, physical activity, employment status, sleep quality and presence of diabetes, cardiovascular disease, and depression were the main determinants of SRH. Those free from cardiovascular disease , rural residents , non-depressed participants  and those with good sleep quality  had almost twice or over the chance of reporting good SRH compared to their counterparts. There were significant associations -and trend- between SRH and levels of inflammatory markers, insulin levels and insulin resistance.Only one third of middle-aged and elderly Chinese assessed their health status as good or very good. Although further longitudinal studies are required to confirm our findings, interventions targeting social inequalities, lifestyle patterns might not only contribute to prevent chronic morbidity but as well to improve populations' perceived health. Self-rated health (SRH) is a simple but comprehensive self-assessment of global  health status in which respondents are asked to rate their overall health, usually in a 5-point scale -3. In prWe aimed to evaluate: 1) the distribution of SRH, among middle-aged and elderly populations in China; 2) the factors that determine SRH in this population; and 3) whether lower SRH is associated with worse levels of biomarkers of cardio-metabolic disease.The current study is part of the "Nutrition and Health of Aging Population in China" study . To provSRH was initially recorded in 5 levels , and then categorized into two categories good (very good and good) and poor  ,13.Socio-demographic variables included age (categorized as 50-59 and 60-70 yrs), gender, geographic region (Beijing/Shanghai), residential area , marital status ) and living status . Socio-economic variables included educational attainment , employment status  and total household income .Lifestyle variables included social activity , smoking habit ; alcohol drinking ("yes "/"no"); physical activity level ; sleBody mass index (BMI) levels were categorized as underweight (<18.5 kg/m2), normal weight (18.5 to <24.0 kg/m2), overweight (24.0 to <28.0 kg/m2) and obese (≥28.0 kg/m2).Medical insurance defined as "yes"/"no" on the basis of benefiting from one type of medical insurance such as social, commercial or governmental paid.Three measurements of blood pressure were taken using an electronic blood pressure monitor and the mean of the last two measurements was used for analysis. Hypertension was defined if either the systolic blood pressure (SBP) is greater than 140 mmHg or diastolic blood pressure (DBP) is greater than 90 mmHg or taking anti-hypertensives. Presence of diabetes was defined as either measured fasting glucose greater than 7.0 mmol/L or being diagnosed with type-2 diabetes or taking anti-diabetic drugs or insulin. Presence of cardiovascular disease (CVD) was defined as the presence of one or more definite manifestations of coronary heart disease or stroke. Metabolic syndrome defined according to the updated National Cholesterol Education Program Adult Treatment Panel III (ATP III) criteria for Asian Americans,22.To measure the presence of clinically relevant depressive symptoms, we used the Center for Epidemiologic Studies Depression Scale (CES-D) , which hIn order to evaluate the association between SRH and cardio-metabolic disorders  we measured a number of biomarkers in our population. Overnight fasting blood samples were collected in tubes containing liquid ethylenediaminetetraacetic acid (EDTA), centrifuged at 4°C, and stored at -80°C until analysis. Total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides (TG), and glucose were measured enzymatically on an automatic analyzer  with reagents purchased from Wako Pure Chemical Industries . Plasma C-reactive protein (CRP) was measured by a particle-enhanced immunoturbidimetric assay  using microparticles coated with anti-human CRP antibodies. Retinol-binding protein 4 (RBP4) was measured by a sandwich ELISA developed in-house, utilizing affinity-chromatography purified polyclonal and monoclonal antibodies generated against recombinant human RBP4, more details can be found elsewhere . Interle) [Plasma adiponectin, resistin and active plasminogen activator inhibitor-1 (PAI-1) concentrations were measured by Luminex xMAPTM Technology  on a Bio-Rad Multiplex Suspension Array System (more details can be found elsewhere . Fasting) .First, we evaluated the distribution of SRH by gender, geographic region and area of residence. Then using the chi-square test, analysis of variance or Wilcoxon rank-sum test, we evaluated the associations between SRH and potential determinants, based on previous literature Table . FactorsAll analyses were repeated stratifying by gender, region and residence one at a time and by stratifying for two of these factors simultaneously.The associations between SRH and biomarkers of cardio-metabolic disease were tested by parametric t-test , or/and non-parametric Wilcoxon rank-sum test. Biomarkers included were: resistin, PAI-1, RBP4, adiponectin, IL-6, CRP, insulin, HOMA-IR, HOMA2-IR, SBP, DBP, HDL cholesterol, LDL cholesterol, total cholesterol and triglycerides. These analyses were performed on the total population and on a sub-sample of participants that were free from CVD, cancer and diabetes.Statistical analyses were performed with the SAS statistical package version 9.1 . Statistical tests were two-sided and at the 5% level of significance.The mean age of the participants was 58.6. There were more women than men (55.7% vs. 44.3%) , almost half of the population reported their health as fair (53%) and the rest as poor or very poor (15%) . Lower prevalence of high physical activity (45.5% vs. 61.4%) and good sleep quality (44.1% vs. 72.5%) and higher prevalence of CVD (14% vs. 2.6%), diabetes (16.9% vs. 7.7%) and depressive symptoms (12.2% vs. 3.6%) was observed among those that reported poor SRH compared to their counterparts  1.89; 95% Confidence Interval (CI)1.47-2.43), being employed , drinking alcohol , having high levels of physical activity  and benefiting from good sleep quality  increased the chance of reporting a good SRH. Furthermore, being free of diabetes , hypertension , CVD  and depression  independent of the other determinants significantly increased the chance of feeling healthier  or gender. Quality of sleep, diabetes and CVD were found significantly associated with SRH for all subgroups. We have also found significant association of depression , hypertension  and physical activity  in most of the subgroups. Among rural residents, drinking alcohol  and among urban residents region of living  was significantly associated with SRH  were observed between the participants with different level of SRH. The level of SRH inversely related to the levels of resistin, CRP, insulin, HOMA-IR, HOMA2-IR, systolic and diastolic blood pressure Table . Trends We found a relatively low level of good SRH among middle-aged and elderly Chinese (32%), which varied substantially by area of residence, lifestyle and socio-economic factors and presence of chronic diseases. Lower levels of SRH were associated with worsened levels of inflammatory markers, blood lipids, insulin and insulin resistance in both the total population and a sub sample free from CVD, diabetes and cancer.Only one third of the elderly Chinese assessed their health as good or very good. Despite lower socio-economic status , rural residents were almost twice as likely to feel healthier compared to urban residents. This could be the consequence of different lifestyle followed by those living in rural areas, characterized by higher levels of physical activity, lower BMI and a higher age of retirement. Furthermore, the prevalence of both CVD  and diabetes  was lower among rural residents  or the coverage of our sample.In previous studies, Yu et al.  by studySimilar to our findings, Leung et al.  by studyWe found a significant association between different lifestyle characteristics and SRH in the current analyses. High levels of physical activity were associated with better SRH. Sleep quality was one of the factors which had the highest level of association with SRH in the study population. Those with good sleep quality were almost twice likely to report good levels of SRH. This result was consistent for all subgroups  of the study population. The effect of smoking and alcohol drinking on SRH could be hampered by the effect of gender as the majority of Chinese smokers and drinkers were men. Nevertheless, we found a significant association of alcohol intake and SRH on the total population and among rural residents. The association however indicated the higher chance of reporting good SRH for those drinking alcohol. This could be due to the lack of information about drinking habits  in the models.Furthermore, socio-economic factors, such as education and employment status, affected mainly urban women. Most of the previous studies ,13,31 inAbsence of CVD, hypertension, diabetes or depression increased the chance of reporting a good level of SRH. Presence of depressive symptoms had a bigger impact on women and urban residents. Suffering from CVD was found consistently associated with SRH levels for all subgroups of gender and area of residence with the strongest association among rural-men as more likely to report poor SRH. The deleterious association of CVD presence on SRH was higher among rural residents and men compared to urban residents and women. Consistent with our findings, by studying 1589 elderly Chinese in Hong Kong, Cheng et al.  found a Lower concentrations of inflammatory biomarkers, plasma insulin and index of insulin resistance were associated with good levels of SRH for the total population and among a subgroup of participants that were free from cancer, CVD and diabetes. Levels of SRH might effectively reveal early deviations from healthy trajectories, making SRH an effective assessment tool to evaluate healthy aging targeting the early prevention of the appearance of chronic disease. To our knowledge, no study has been conducted on Chinese in mainland China to examine the association between cardio-metabolic biomarkers and SRH. Only few studies investigated this aspect. For instance, consistent with our findings, Tomten et al.  studied This is as well the first study to ever evaluate the associations between SRH and inflammatory markers and measures of insulin resistance in any population and the first study to evaluate the levels and distribution of SRH, its determinants and its associations with biomarkers of cardio-metabolic disease among Chinese populations from different geographical areas and residence.We considered the effect of comprehensive measurements of lifestyle, socio-demographic factors and presence of disease in the analysis. The extrapolation of our findings is somewhat limited due to the cross-sectional nature of our data and further longitudinal studies are required to confirm our findings. Additionally no clinical diagnostic was carried out to validate the result and the severity of the diseases. Also the functional and/or cognitive level of participants, which has been previously found to be associated with SRH , was notIn general, we have found relatively low levels of good SRH in our population alongside with substantial impacts of area of residence, sleep quality and chronic diseases on SRH. Good SRH was associated with lower levels of inflammatory markers, insulin levels and insulin resistance, which might present SRH as a potential assessment tool to evaluate early deviations from health. Although further longitudinal studies are required to confirm our findings, interventions targeting social inequalities, sleep and physical activity patterns might not only contribute to prevent chronic morbidity but also to improve populations' perceived health.BMI: body mass index; CI: confidence interval; CVD: cardiovascular disease; DBP: diastolic blood pressure; HDL: high-density lipoprotein; LDL: low-density lipoprotein; MetS: metabolic syndrome; OR: odds ratio; SBP: systolic blood pressure; SD: standard deviation; SRH: self-rated health; TG: triglycerides; TCH: total cholesterol; RBP4: retinol-binding protein 4; ADIPO: adiponectin; IL-6: interleukin-6; PAI-1: plasminogen activator inhibitor-1; CRP: C-reactive protein; ANOVA: analysis of varianceThe authors declare that they have no competing interests.NHM performed the statistical analysis, interpretation of data and sequence alignment and drafted the manuscript. AP participated in design of the study, data collection and manuscript revision. XY, JW, QQ, YL, HL, ZY participated in the design of the study and data collection. XL carried out in design of the study and supervised the study progress in china. OHF conceived the study and participated in the analysis, drafting and revising the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The exosome complex is an essential RNA 3′-end processing and degradation machinery. In archaeal organisms, the exosome consists of a catalytic ring and an RNA-binding ring, both of which were previously reported to assume three-fold symmetry.Sulfolobus solfataricus archaeal exosome structure in which the three-fold symmetry is broken due to combined rigid body and thermal motions mainly within the RNA-binding ring. Since increased conformational flexibility was also observed in the RNA-binding ring of the related bacterial PNPase, we speculate that this may reflect an evolutionarily conserved mechanism to accommodate diverse RNA substrates for degradation.Here we report an asymmetric 2.9 Å This study clearly shows the dynamic structures within the RNA-binding domains, which provides additional insights on mechanism of asymmetric RNA binding and processing. The exosome and its related bacterial polynucleotide phosphorylase (PNPase) represent a class of conserved multi-subunit protein complexes responsible for the 3′-to-5′ processing and degradation of RNA The function and architecture of the exosome is conserved among all three kingdoms of life. Its bacterial counterpart, PNPase, is an 81 KDa polypeptide that trimerizes to form a ∼240 KDa complex. The primary sequence of the PNPase contains two domains homologous to RNase PH, followed by RNA-binding KH and S1 domains The structure and enzymatic mechanism of the archaeal exosome have been extensively studied. Published structural work revealed that the archaeal exosomes adopt a three-fold symmetric structure, where a trimeric RNA-binding ring packs on top of the catalytic ring consisting of a trimer of Rrp41/Rrp42 heterodimers. A large “processing chamber” was found inside the catalytic ring, where three identical phosphorolytic active sites reside. RNA substrate likely gains access to the “processing chamber” from the side of the RNA-binding ring through a narrow pore at the neck region, which only allows the passage of a single-stranded (ss) RNA Sulfolobus solfataricus exosome. We observed that while the catalytic ring of the S. solfataricus exosome adopts a three-fold symmetric structure, its RNA-binding ring displays considerable rigid body and thermal motions, which broke its internal symmetry. Since conformational flexibility was also observed in the RNA-binding ring of the bacterial PNPase To further understand the RNA recruitment mechanism of the exosome family of macromolecular machines, we determined the crystal structure of the archaeal Sulfolobus solfataricus exosome by co-expressing all four exosome subunits from a polycistronic construct in E. coliWhen we started the work, studies had shown that all four eukaryotic exosome homologous proteins, namely Rrp41, Rrp42, Rrp4, and Csl4, are present in the purified archaeal exosome complex 43− or in the presence of 20 mM SO42− (data not shown).To demonstrate the phosphorylytic RNase activity in our purified archaeal exosome, we incubated the purified exosome with a 254-nt RNA substrate flanked by a strong 5′-tertiary RNA structure (the hepatitis delta virus ribozyme) and a 161-nt flexible 3′ tail under multiple turnover conditions. As shown in Sulfolobus solfataricus exosome crystals.We obtained the crystals of the four-protein component archaeal exosome complex grown in ammonium sulfate and polyethylene glycol 4000 (PEG 4000). The crystals diffracted X-rays to 2.4 Å resolution at a synchrotron radiation source. Data processing by HKL2000 We reasoned that the problematic diffraction pattern from the four-subunit exosome crystals was due to the presence of a sub-stoichiometric amount of Csl4 protein. To generate homogeneous exosome samples, the Csl4 gene was deleted from the co-expression cassette and biochemically, the resulting Rrp4-exosome isoform behaved similarly as the four-subunit complex. We independently crystallized the complex under high PEG conditions similar to that reported by the Conti group S. solfataricus exosome Rrp4-isoform structure by molecular replacement using the Rrp41/Rrp42 heterodimer in the S. solfataricus exosome catalytic core (PDB code: 2BR2) as the search model work/Rfree were within the acceptable range of 26.7%/28.9%.We solved the S. solfataricus Rrp4-exosome crystal structure  in the RNA-binding ring of our structure, however, deviate significantly from their three-fold symmetric positions as reported previously . When onBesides conformational heterogeneity caused by the hinge motion, the Rrp4 trimer also exhibits distinct temperature factor distributions, which reflect the RNA binding ring's inherent conformational flexibilities. TLS refinement followed by thermal ellipsoids analysis revealed unique thermal motions in Chain I copy of Rrp4 . AlthougS. solfataricus exosome RNA-binding ring differs dramatically from what was seen in the A. fulgidus structure mostly due to rigid body rearrangement of the KH, S1, and NT domain locations S. solfataricus RNA-binding ring adopts a distinct conformation and thermal motion distribution pattern. As a result, the previously assumed three-fold symmetric subunit arrangement is broken. Interestingly, it was found that significantly higher conformational flexibility and thermal motions are present in the RNA-binding ring of the crystal structures of the exosome-related PNPases from mesophilic bacteria, Escherichia coliStreptomyces antibioticusE. coli PNPase is so flexible that it could not be successfully traced from the electron density map Previous structural studies of the archaeal exosome from three different species all reported perfect three-fold symmetric subunit arrangements within this RNA processing machine. An interesting observation from these studies was that the RNA-binding ring displays large conformational differences among different species. For example, the conformation of the S. solfataricus exosome, but can later be gradually degraded by the exosome, albeit at much slower rate  The conformational flexibility may play a passive role in accommodating the binding of diverse RNA substrates before threading the RNA into the catalytic ring. The fact that each of the three Rrp4 subunits possesses distinct thermal and conformational characteristics suggests that the exosome might respond to the substrate by activating one of the three Rrp4 subunits. It is also possible that the substrate binding is a cooperative event that involves more than one Rrp4 subunit. (2) On the other hand, the increased thermal motion and flexibility in this region may further play an active role in unzipping the secondary structures of the RNA substrates so that the RNA is ‘ready’ to pass through the narrow neck region of the exosome, which can only accommodate a single-stranded RNA. The RNA-binding ring therefore resembles an RNA helicase in its RNA-unwinding function. Consistent with this hypothesis, we observed that a strong tertiary structure, such as the HDV ribozyme, initially stalls the action of the wer rate .Sulfolobus solfataricus. We designed a polycistronic construct to promote exosome assembly by co-expressing four subunits in E. coli simultaneously. This was inspired by the observation that the three Rrp genes are arranged as a superoperon (in the order of Rrp4-Rrp41-Rrp42) in the archaeal genomes 6-tag and a Tobacco Etch Virus (TEV) protease cleavage site were engineered into the N-terminus of Rrp4 to facilitate purification.Genes encoding Rrp4, Rrp41, Rrp42, and Csl4 have been cloned from the genomic DNA of the archaeal hyperthermophile E.coli, the supernatant of the cell lysate was incubated at 75° for half an hour and precipitated using centrifugation to efficiently denature and remove E. coli proteins. Target proteins were then purified using nickel affinity chromatography using supernatant from the heat treatment. All four exosome subunits were present in the eluate in roughly predicted stoichiometry. Purified proteins were then dialyzed to a buffer containing 25 mM Tris-HCl pH 8.0 and 100 mM NaCl, incubated with TEV protease to remove the His6-tag, and further purified to homogeneity on an anion exchange chromatography (Mono Q column). Alternatively, the complex can by purified using size exclusion chromatography (Suprose 6 column). We confirmed the identity of the four archaeal proteins in the purified exosome by separation on SDS polyacrylamide gel (SDS-PAGE), followed by in-gel protease digestion, electrospray mass spectrometry, and data base matching (data not shown).After overexpression in in vitro T7 RNA polymerase transcription reaction and purified following standard protocols as previously described 43−). Control experiments were carried out in the same buffer in the absence of PO43− or in the presence of 20 mM SO43−.The 254-nt RNA substrate containing the hepatitis delta virus ribozyme followed by a 161-nt 3′-tail was generated from 2, and 25 mM (NH4)2SO4), and crystallized by hanging vapor diffusion against a well solution containing 30% PEG4000 with 100 mM Tris-HCl pH 8.6, 200 mM Mg(Ac)2. Crystals appeared after two months incubation at room temperature among heavy precipitations. The crystals were soaked in the well solution plus 10 mM NaSO4 for 10 minutes before flash-frozen in liquid nitrogen. A complete data set to 2.9 Å was collected at 90K from CHESS beamline A1. The crystal belongs to C2 space group with unit cell dimensions of 151 Å×145 Å×97 Å, and γ = 93.8°, as indexed and scaled by HKL2000 S. solfataricus exosome (PDB code: 2BR2) o-Fc difference map. Initially, strict three-fold non-crystallographic symmetry (NCS) was applied to refine the catalytic ring structure (Rrp41/42 heterotrimer) but was dropped when refinement can no longer improve the model and Rfree. Only two of the three Rrp4 subunits can be correctly placed by molecular replacement in PHASER using the published Rrp4 structure (PDB code 2JE6) work/Rfree to 0.31/0.34 and positioned all three Rrp4 proteins into density map. At this stage the, Rwork/Rfree stopped dropping due to over refinement in some of the subunits and under refinement in others. Therefore, we treated each of the nine subunits separately and independently subject them to energy minimization, simulated annealing, grouped B-factor refinement in CNS work/Rfree to 0.29/0.31. We then defined a total of 15 translation-liberation-screw (TLS) groups in the entire structure: three of each Rrp41, Rrp42, N-ter, S1 and KH domain of Rrp4 subunit, to be used in TLS refinement in Refmac5 which ultimately reduced refinement statistics to the final Rwork/Rfree = 0.268/0.289 68/0.289 , Fig. 1d


Escherichia coli associated with the Moco biosynthetic operon controls gene expression in response to Moco production. In addition, we provide evidence indicating that this conserved RNA discriminates against closely related analogues of Moco. These results, together with extensive phylogenetic conservation and typical gene control structures near some examples, indicate that representatives of this structured RNA represent a novel class of riboswitches that sense Moco. Furthermore, we identify variants of this RNA that are likely to be triggered by the related tungsten cofactor (Tuco), which carries tungsten in place of molybdenum as the metal constituent.We have identified a highly conserved RNA motif located upstream of genes encoding molybdate transporters, molybdenum cofactor (Moco) biosynthesis enzymes, and proteins that utilize Moco as a coenzyme. Bioinformatics searches have identified 176 representatives in γ-Proteobacteria, δ-Proteobacteria, Clostridia, Actinobacteria, Deinococcus-Thermus species and DNAs from environmental samples. Using genetic assays, we demonstrate that a Moco RNA in  Riboswitches are structured RNA domains that selectively bind metabolites or metal ions and function as gene control elements . RiboswiRiboswitches make excellent targets for discovery using bioinformatics searches. For example, the CMfinder comparative genomics pipeline  form the secondary structure architecture of Moco RNAs. Within this conserved secondary structure is a GNRA tetraloop  and a teE. coli Moco RNA upstream of the moaABCDE operon  where Moco motifs are located upstream of both the MPT biosynthetic operon and a gene predicted to encode MPT oxidoreductase. The Moco motif is also found upstream of different arrangements of genes for Moco biosynthesis enzymes, for high-affinity molybdate transporters, and for enzymes that use Moco to catalyse reactions. In general, these genomic contexts point to Moco or a biosynthetic intermediate of this cofactor as the potential ligand for this riboswitch candidate. Several of the known riboswitch classes sense and respond to other common coenzymes, and therefore we considered Moco as the most likely ligand for this riboswitch candidate.In our effort to establish the function of Moco RNAs, we considered the functions of genes in the neighbouring genomic locations that carry representatives of this conserved RNA. In γ-Proteobacteria, including E operon , which eE operon . In some complex . There aE. coli there are five known operons involved in Moco metabolism: moa, mob, mod, moe and mog  by MobA , the predicted ribosome binding site, and AUG translation start codon of the moaA ORF. The RNA also carries two additional G residues at the 5′-terminus to facilitate production by in vitro transcription. A trace amount of 5′32P-labelled 138 moaA RNA was subjected to in-line probing analysis , the resulting pattern of RNA cleavage products  generateIf Moco RNAs indeed serve as aptamer domains for riboswitches, the in-line probing results suggest that the P2 stem and the junctions carrying several of the highly conserved nucleotides might undergo a conformational change to form a ligand binding pocket. This would be consistent with the behaviour of other known riboswitch aptamers, some of which are observed by in-line probing assays to undergo substantial changes in structure on addition of the target metabolite  moaA 5′ UTR. This genome segment encodes a 149 nt RNA (termed 149 moaA) that begins 9 nt upstream of the 5′ beginning of P1 and extends 21 nt beyond the 3′ end of P1 into the moaA coding region. Also, two variant DNAs were generated that express the 149 moaA RNA with mutations that disrupt (M1) and subsequently restore (M2) base pairing in the P3 stem  results in derepression of gene expression. In contrast, altering P3 sequences using additional mutations that restore base pairing (M2) also restores gene repression on arabinose-induction of Moco biosynthesis. The disruption of gene control and the return to WT reporter gene expression levels exhibited by these variants indicates that the Moco RNA is a gene control element that requires its conserved secondary structure for activity.E. coli cells via a high-affinity protein-dependent ABC transporter that is encoded by the mod operon  medium  medium , which cpression . In contmoaA–reporter fusion construct in the transformant carrying the modC deletion becomes derepressed. As noted above, tungsten can enter cells and replace molybdenum as the metal ion component of the cofactor. However, if the 149 moaA RNA functions as a metabolite-sensing riboswitch (e.g. Moco), it has the ability to discriminate between a compound coordinated to molybdenum and the analogous compound coordinated with tungsten. In all cases, the reporter fusion constructs carrying the disruptive (M1) and compensatory (M2) mutations yield the expected gene expression profiles.Interestingly, when cells are grown in LB supplemented with 10 mM sodium tungstate, the WT 149 moaA knockout examined earlier , and this activity is required for regulation of gene expression by the 149 moaA RNA.The gene expression characteristics of the Δeviously , which dFor numerous riboswitch classes, the ligand sensed by the riboswitch aptamer is the final coenzyme or metabolic product in the biosynthetic pathway being regulated . Moco isE. coli, before Moco can be inserted into a protein it must be further modified by MobA . However, the groupings do not break down strictly along phylogenetic divisions, which would be consistent with the propagation of structural variants of a riboswitch aptamer that recognize the same ligand. Therefore, we suspected that there might be some biochemical reason for the structural distinctions and their particular phylogenetic distribution.Haemophilus ducreyi and Hyphomonas neptunium, our bioinformatics searches for coenzyme-related genes are unlikely to be comprehensive. Furthermore, the P3-lacking RNAs are almost exclusively associated with genes encoding enzymes associated with MPT biosynthesis (MPT is a precursor to both Tuco and Moco biosynthesis) or with genes encoding enzymes that are known or predicted to use Tuco as a coenzyme.Interestingly, the two structural types of Moco RNAs exhibit near perfect correspondence with the utilization of Moco and Tuco among bacteria , suggestPelobacter carbinolicus, which has a P3-lacking RNA associated with a predicted molybdate transporter (a). However, a member of this transporter class is not able to discriminate against tungstate, and therefore the transporter in this organism might actually be important for Tuco biosynthesis. Even in organisms that carry both structural variations of Moco RNAs, the segregation of the P3-inclusive and P3-lacking RNAs to Moco and Tuco metabolic processes, respectively, is largely is maintained. An apparent exception is found with Moorella thermoacetica (b), where a P3-lacking RNA is associated with a gene (nirB) whose function is predicted to be related to Moco metabolism. However, this gene resides in an operon with a predicted Tuco-related gene (see the legend to S. wolfei (c) that are associated with genes that encode putative Moco-utilizing sulphite oxidase enzymes. If the gene products indeed use Moco, perhaps expression of these genes is repressed when Tuco concentrations increase if the cell has an alternative biosynthetic pathway that uses Tuco. Also, we cannot rule out the possibility that the gene products might actually use Tuco instead of Moco.An apparent exception to the segregation of Moco RNA types with Moco and Tuco metabolism is evident for oacetica , b, wher. wolfei , c that E. coli Moco RNA examined in this study, but this change completely disrupted gene regulation under all growth conditions tested (data not shown). Presumably, this deletion alone is insufficient to switch specificity of Moco RNAs from Moco to Tuco.Regardless, the striking segregation of the two types of Moco RNAs could be explained by the existence of two Moco RNA types, differing by the presence or absence of the P3 stem, that selectively sense Moco or Tuco respectively. We have deleted the P3 stem from the S. wolfei. The genes in this operon are predicted to encode proteins involved in MPT synthesis, molybdate or tungstate transport, and other genes of unknown function. All three RNAs in this series lack P3 stems. In addition, we observe putative intrinsic transcription terminators following the first two RNA motifs, and a long gap between the third motif and the first ORF in the operon. If each riboswitch indeed functions via a separate expression platform, and if the absence of a P3 stem is indicative of Tuco binding, then the triple arrangement might allow cells to be exceptionally responsive to small changes in Tuco concentration, with greater ‘digital’ gene control character compared with tandem riboswitches describe previously  and enabling (sufficient levels of molybdate sensed by ModE) Moco production are present in the cell. If the Moco RNA indeed is a riboswitch, it adds an additional layer of regulation. A Moco-sensing riboswitch would ensure that if there are sufficient levels of Moco already present, as indicated by free Moco binding to the moaABCDE transcript, then the Moco biosynthetic proteins are not made. This system allows for a rapid yet efficient response to the changing demand for Moco within the cell.In moaA RNA exhibits gene control functions that are dependent on its secondary structure. However, the instability of Moco and its metabolic intermediates precludes us from demonstrating direct binding of these coenzymes to the RNA with commonly used techniques such as in-line probing, equilibrium dialysis or fluorescence-based assays. These latter experiments are typically used to prove that ligand binding occurs in the complete absence of protein factors.The experimental and bioinformatics data presented herein is consistent with our hypothesis that RNAs conforming to the Moco RNA motif are most likely serving as Moco- or Tuco-sensing riboswitch aptamers. The RNAs have genomic distributions that are consistent with riboswitch function, and the 149 E. coli , and the distributions of these motif types correlate with an organism's use of Moco, Tuco, or both  ; d-glucose, 0.2% (w/v). Glycerol, 0.2% (w/v), is added to the medium when arabinose supplementation is applied (The ΔlacX74)  was obtaΔlacX74)  were obtmoaA RNA construct used for in-line probing was prepared by in vitro transcription using a template generated by successive PCR amplification reactions. The intergenic region upstream of moaA was amplified by PCR from E. coli K12 genomic DNA using the primers 5′-GAATTCCCTGGAGTCAGATTATCCGC and 5′-CTGGATCCAGTTGTGAAGCCATGTACAC. Following digestion by EcoRI and BamHI, the PCR product was cloned into pRS414 and the integrity of the resulting plasmid was confirmed by DNA sequencing. This plasmid was used as the template for PCR amplification of the DNA construct encoding the 138 moaA construct (including the GG addition corresponding to the 5′ terminus of the RNA transcript) where the primer for the non-template strand included the promoter sequence for T7 RNA polymerase. RNA molecules were prepared by transcription in vitro using T7 RNA polymerase and purified using denaturing PAGE as described previously  and 20 pmol was radiolabelled with [γ-32P]-ATP using T4 polynucleotide kinase (New England Biolabs) following protocols supplied by the manufacturers. In-line probing reactions using radiolabelled RNAs purified by PAGE were assembled as previously described  and flanking gene-specific primers . The kanamycin cassette was then eliminated by site-specific recombination by using the FLP plasmid pCP20. The resulting kanamycin sensitive transformants were verified by PCR with the same primers used for the pre-FLP verification.The resulting ∼1.4 kb long PCR products, containing a kanamycin resistance cassette flanked by FLP recognition target sites and identical 50 nt sequence identities to adjacent chromosomal sequences, were purified and subjected to DpnI digestion. DJ480 cells were transformed with the Red helper plasmid, pKD46, encoding the Red recombinase from phage λ that promotes homologous recombination. Transformants were selected for ampicillin resistance. DJ480/pKD46 cells grown with lac UV5 promoter lacking the operator binding site was achieved as follows. The nucleotide sequence from 1 to 145 relative to the S1 transcription start site for the E. coli moaABCDE operon   and the appropriate mutagenic DNA primers. All mutations were confirmed by DNA sequencing.Preparation of a Moco RNA–reporter fusion construct driven by the constitutively active E operon  was amplmoaABCDE operon was cloned into the pBAD33 plasmid (moaABCDE fragment was amplified by PCR from E. coli K12 using primers 5′-CTAGGTACCTCTGGAAAGGTGTACATGGCTTCACAAC and 5′-TAGAAGCTTAACTACCAGCGTTTTGCCGCCTGCTG. The PCR product was purified by agarose gel electrophoresis and subjected to KpnI and HindIII digestion, as was pBAD33. The digested fragment was then ligated into pBAD33 downstream of the arabinose-inducible promoter and transformed into NEB-5α cells. Transformants were selected for chloramphenicol resistance and the fusion was confirmed by DNA sequencing.The moaA, mogA, modC, moeA and mobA knockout DJ480 strains were transformed simultaneously with both the lac UV5–Moco motif–lacZ fusion pRS414 plasmid containing WT or mutant 149 moaA–reporter fusions and the moaABCDE pBAD33 overexpression plasmid. Transformants were selected for both ampicillin and chloramphenicol resistance.The lacZ in the WT and mutant reporter strains of E. coli, the cells were grown in a 14 ml culture vial with shaking overnight at 37°C in 3 ml of LB with supplementation as indicated. The cells were then diluted to an OD600 of 0.05 in 150 μl of LB in 96-well plates and grown to an A600 between 0.5 and 0.7 with shaking at 37°C, and then harvested for gene expression experiments. β-Galactosidase activity was assayed using a 96-well microplate protocol described elsewhere (To measure gene expression from blast to search for genes homologous to tupA and tupB. These genes have been shown in Eubacterium acidaminophilum to encode transporters specific for tungstate (Pelobacter carbinolicus and Desulfotalea psychrophilia were assigned based on the presence of wtpA homologues, which have been shown to be distinct but selective transporters of tungstate (The assignment of Moco and Tuco metabolism to various organisms and the assignment of Moco RNA motif types to genes involved in three processes (MPT biosynthesis, Moco biosynthesis or use, and Tuco biosynthesis or use;