In the fluorescent spotlight: Global and local conformational changes of small catalytic RNAs

RNA is a ubiquitous biopolymer that performs a multitude of essential cellular functions involving the maintenance, transfer, and processing of genetic information. RNA is unique in that it can carry both genetic information and catalytic function. Its secondary structure domains, which fold stably and independently, assemble hierarchically into modular tertiary structures. Studies of these folding events are key to understanding how catalytic RNAs (ribozymes) are able to position reaction components for site-specific chemistry. We have made use of fluorescence techniques to monitor the rates and free energies of folding of the small hairpin and hepatitis delta virus (HDV) ribozymes, found in satellite RNAs of plant and the human hepatitis B viruses, respectively. In particular, fluorescence resonance energy transfer (FRET) has been employed to monitor global conformational changes, and 2-aminopurine fluorescence quenching to probe for local structural rearrangements. In this review we illuminate what we have learned about the reaction pathways of the hairpin and HDV ribozymes, and how our results have complemented other biochemical and biophysical investigations. The structural transitions observed in these two small catalytic RNAs are likely to be found in many other biological RNAs, and the described fluorescence techniques promise to be broadly applicable. © 2002 Wiley Periodicals, Inc. Biopoly (Nucleic Acid Sci) 61: 224–241, 2002Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34325/1/10144_ftp.pd

2 Terbium(III) Footprinting as a Probe of RNA Structure and Metal Binding Sites

Introduction Cations play a pivotal role in RNA structure and function. A functional RNA tertiary structure is stabilized by metal ions that neutralize and, in the case of multivalent ions, bridge the negatively charged phosphoribose backbon

Investigation of the mechanism of chromium removal in (3-aminopropyl)trimethoxysilane functionalized mesoporous silica

We are proposed that a possible mechanism for Cr(VI) removal by functionalized mesoporous silica. Mesoporous silica was functionalized with (3-aminopropyl)trimethoxysilane (APTMS) using the post-synthesis grafting method. The synthesized materials were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), N-2 adsorption-desorption analysis, Fourier-transform infrared (FT-IR), thermogravimetric analyses (TGA), and X-ray photoelectron spectroscopy (XPS) to confirm the pore structure and functionalization of amine groups, and were subsequently used as adsorbents for the removal of Cr(VI) from aqueous solution. As the concentration of APTMS increases from 0.01 M to 0.25 M, the surface area of mesoporous silica decreases from 857.9 m(2)/g to 402.6 m(2)/g. In contrast, Cr(VI) uptake increases from 36.95 mg/g to 83.50 mg/g. This indicates that the enhanced Cr(VI) removal was primarily due to the activity of functional groups. It is thought that the optimum concentration of APTMS for functionalization is approximately 0.05 M. According to XPS data, NH3+ and protonated NH2 from APTMS adsorbed anionic Cr(VI) by electrostatic interaction and changed the solution pH. Equilibrium data are well fitted by Temkin and Sips isotherms. This research shows promising results for the application of amino functionalized mesoporous silica as an adsorbent to removal Cr(VI) from aqueous solution

Probing RNA Structure and Metal‐Binding Sites Using Terbium(III) Footprinting

The function of an RNA molecule is determined by its overall secondary and tertiary structure. The tertiary structure is facilitated and stabilized by the interaction with metal ions. The current chapter offers a detailed protocol on the use of the lanthanide metal ion terbium(III) as a powerful probe of RNA structure and metal‐binding properties. When incubating RNA with low (micromolar) concentrations of terbium(III), specific backbone scission by partially deprotonated aqueous terbium(III) complexes can be used to detect high‐affinity metal‐binding sites, while incubation with high (millimolar) terbium(III) concentrations cleaves the RNA backbone preferentially at structurally accessible regions, providing a footprint of the RNA secondary and tertiary structure.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/153092/1/cpnc0608.pd

Local conformational changes in the catalytic core of the trans-acting hepatitis delta virus ribozyme accompany catalysis. Biochemistry

ABSTRACT: The hepatitis delta virus (HDV) is a human pathogen and satellite RNA of the hepatitis B virus. It utilizes a self-cleaving catalytic RNA motif to process multimeric intermediates in the doublerolling circle replication of its genome. Previous kinetic analyses have suggested that a particular cytosine residue (C 75 ) with a pK a close to neutrality acts as a general acid or base in cleavage chemistry. The crystal structure of the product form of a cis-acting HDV ribozyme shows this residue positioned close to the 5′-OH leaving group of the reaction by a trefoil turn in the RNA backbone. By modifying G 76 of the trefoil turn of a synthetic trans-cleaving HDV ribozyme to the fluorescent 2-aminopurine (AP), we can directly monitor local conformational changes in the catalytic core. In the ribozyme-substrate complex (precursor), AP fluorescence is strongly quenched, suggesting that AP 76 is stacked with other bases and that the trefoil turn is not formed. In contrast, formation of the product complex upon substrate cleavage or direct product binding results in a significant increase in fluorescence, consistent with AP 76 becoming unstacked and solvent-exposed as evidenced in the trefoil turn. Using AP fluorescence and fluorescence resonance energy transfer (FRET) in concert, we demonstrate that this local conformational change in the trefoil turn is kinetically coincidental with a previously observed global structural change of the ribozyme. Our data show that, at least in the trans-acting HDV ribozyme, C 75 becomes positioned for reaction chemistry only along the trajectory from precursor to product. The hepatitis delta virus ribozyme is among a class of small endonucleolytic RNAs that catalyze a reversible selfcleavage reaction necessary for the replication and propagation of their satellite RNA genomes. Specifically, the hepatitis delta virus ribozyme is a unique RNA motif found in the human hepatitis delta virus (HDV) 1 (1). HDV is a satellite of the hepatitis B virus (HBV); coinfection of HDV and HBV results in intensification of the disease symptoms associated with the hepatitis B virus (2). The small RNA genome of HDV replicates through a double-rolling circle mechanism, whereby multimeric units of genomic and antigenomic RNA strands are produced, followed by self-cleavage and ligation into circular monomers (1, 3). Self-cleavage activity in the genomic and antigenomic RNAs resides within continuous 85-nucleotide sequences that both form a nearly identical secondary structure consisting of a nested double pseudoknot (4, 5). The genomic and antigenomic forms of the HDV ribozyme catalyze self-cleavage by a transesterification reaction, which requires deprotonation of the adjacent 2′-OH group and its nucleophilic attack on the scissile phosphate, resulting in formation of 2′,3′-cyclic phosphate and 5′-OH termini (5). The reaction mechanism of the HDV ribozyme has been extensively studied. The crystal structure of the self-cleaved genomic ribozyme reveals that the base cytosine 75 (C 75 ) is situated in the active site cleft and, thus, in the proximity of the 5′-OH leaving group Several biochemical and mutagenesis studies support the idea that C 75 in the genomic ribozyme and the corresponding RC 76 (R used to distinguish antigenomic numbering) in the antigenomic ribozyme are involved in catalysis (7-10). The pH dependence of self-cleavage (or cis cleavage) by the HDV ribozyme reveals a macroscopic apparent pK a that approaches neutrality. In a widely accepted model, this pK a reflects the ionization equilibrium of N3 in C 75 which therefore is strongly shifted in the folded ribozyme compared to that in the free base (pK a ≈ 4.2). A decrease in this pK a for selfcleavage of an antigenomic ribozyme with an RC 76 A mutation was observed, consistent with A substituting for C in this position to act as a general base catalyst (8). However, the pH profile of the genomic ribozyme in the presence of 1 M NaCl and 1-100 mM EDTA favors a model where C 75 acts as a general acid during catalysi

Faecal elastase 1 levels in premature and full term infants

Background: Determination of faecal elastase 1 (FE1) is a simple, relatively inexpensive, non-invasive, highly specific and sensitive test for determining pancreatic function. Secretion of pancreatic enzymes varies during infancy, but there are almost no specific data on the ontogeny of elastase 1 in human babies. Aim: To study FE1 levels in preterm and term babies, and to determine the possible effect of gestational and postconceptual age on these levels. Methods: Serial stool samples were collected and tested for FE1 level from 77 premature and full term infants. FE1 levels were determined by a commercially available enzyme linked immunosorbent assay (ELISA) kit. Results: A total of 232 stool samples were collected from 77 neonates. The FE1 level measured in the first stool sample (meconium) was below normal (200 µg/g stool) in all samples regardless of gestational age. Sixty three neonates had at least two samples tested for FE1 level. The mean (SD) level of FE1 in sample 1 was 45.9 (51.1) µg/g stool and was significantly (p < 0.001) lower than in sample 2 (243.0 (164.9) µg/g stool). The lower the gestational age of the newborn, the more time it took for FE1 to reach normal levels. Conclusions: FE1 levels in meconium are low, and studies in meconium should be avoided if pancreatic sufficiency is to be determined. FE1 reaches normal levels by day 3 in term newborns and by 2 weeks in infants born before 28 weeks gestation. Normal levels are reached sooner in infants of more advanced gestational age who start enteral feeding earlier

Efficacy of lamivudine for the treatment of hepatitis B virus infection after liver transplantation in children

Background. There is at present very little information about hepatitis B virus (HBV) infection in children after liver transplantation. This is the first study to assess the safety and efficacy of lamivudine in this patient population. Methods. We describe three children aged 5-14 years who underwent liver transplantation for fulminant hepatitis A, hyperoxaluria, and cystic fibrosis. Despite adequate immunoprophylaxis, two of the children who were serum hepatitis B surface antigen-positive before transplantation (HBV DNA-negative by hybridization) had a reactivation of the disease, and one had a de novo HBV infection, at 12-18 months after transplantation. Lamivudine 3 mg/kg was administered on a compassionate-use basis for 14-36 months. Results. After 1 month of therapy, HBV DNA disappeared from the serum in all patients by hybridization and in two patients by polymerase chain reaction. In all three children, alanine transaminase levels normalized. One child developed lamivudine resistance after 22 months with no evidence of hepatic decompensation. Repeated liver histological studies revealed progression of hepatic fibrosis in one child. All children remained serum hepatitis B surface antigen-and hepatitis B e antigen-positive. No adverse effects of the drug were noted. Conclusion. Lamivudine is beneficial and well tolerated in children with HBV infection after liver transplantation