Structural Basis for tRNA Mimicry by a Bacterial Y RNA

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Factors Affecting Deoxycholate Inactivation and Mg++ Reactivation of Bacillus megaterium KM Membrane Nicotinamide Adenine Dinucleotide (Reduced Form) Oxidase

Decay of the reduced nicotinamide adenine dinucleotide oxidase of Bacillus megaterium KM membranes was prevented by storage in 10% (v/v) glycerol or 0.4% bovine serum albumin. Differential rates of solubilization of components of the oxidase system by 0.4% deoxycholate was demonstrable at 4 C. The amount of Mg(++) necessary for maximal oxidase reactivation increased with increasing amounts of deoxycholate-inactivated oxidase. Mg(++) activation of deoxycholate-inactivated oxidase was partially temperature-dependent. Maximal activation was observed at 37 C, but only partial activation took place at 4 C. A small amount of deoxycholate was required for Mg(++) activation of deoxycholate-inactivated oxidase. Two pH optima were found for Mg(++) activation of deoxycholate-inactivated oxidase, pH 5.3 and 7.3. Significant amounts of activation of the inactive oxidase occurred in the absence of Mg(++) with an optimum at pH 5.0, with essentially no Mg(++)-independent activation demonstrable at pH 7.0.     

Genes for two small cytoplasmic Ro RNAs are adjacent and appear to be single-copy in the human genome

Anti-Ro autoantibodies precipitate several small cytoplasmic ribonucleoproteins from mammalian cells. The RNA components of these particles, designated hY1-hY5 in human cells and mY1 and mY2 in mouse cells, are about 100 nucleotides long. We have analyzed a genomic clone that appears to contain true RNA-coding regions for two of the human Ro RNAs, hY1 and hY3. These RNAs exhibit many sequence and secondary structure homologies, both with each other and with the recently sequenced hY5 RNA. The hY2 RNA is a slightly truncated form of hY1; several shorter versions of hY3 are also detected in cell extracts and immunoprecipitates. The human hY1 and hY3 genes cross-hybridize with the mouse Ro RNAs, mY1 and mY2, respectively; we show that the mouse Ro RNAs are exclusively contained in Ro particles. The genes for hY1 and hY3 are transcribed in vitro by RNA polymerase III. In contrast with all other mammalian class III genes described, they appear to be present as single copies in the human genome.     

6-Substituted quinolines as RORγt inverse agonists

We identified 6-substituted quinolines as modulators of the retinoic acid receptor-related orphan receptor gamma t (RORγt). The synthesis of this class of RORγt modulators is reported, and optimization of the substituents at the quinoline 6-position that produced compounds with high affinity for the receptor is detailed. This effort identified molecules that act as potent, full inverse agonists in a RORγt-driven cell-based reporter assay. The X-ray crystal structures of two full inverse agonists from this chemical series bound to the RORγt ligand binding domain are disclosed, and we highlight the interaction of a hydrogen-bond acceptor on the 6-position substituent of the inverse agonist with Glu379:NH as a conserved binding contact.     

Competition between the Rex1 exonuclease and the La protein affects both Trf4p-mediated RNA quality control and pre-tRNA maturation

Although nascent noncoding RNAs can undergo maturation to functional RNAs or degradation by quality control pathways, the events that influence the choice of pathway are not understood. We report that the targeting of pre-tRNAs and certain other noncoding RNAs for decay by the TRAMP pathway is strongly influenced by competition between the La protein and the Rex1 exonuclease for access to their 3' ends. The La protein binds the 3' ends of many nascent noncoding RNAs, protecting them from exonucleases. We demonstrate that unspliced, end-matured, partially aminoacylated pre-tRNAs accumulate in yeast lacking the TRAMP subunit Trf4p, indicating that these pre-tRNAs normally undergo decay. By comparing RNA extracted from wild-type and mutant yeast strains, we show that Rex1p is the major exonuclease involved in pre-tRNA trailer trimming and may also function in nuclear CCA turnover. As the accumulation of end-matured pre-tRNAs in trf4Delta cells requires Rex1p, these pre-tRNAs are formed by exonucleolytic trimming. Accumulation of truncated forms of 5S rRNA and SRP RNA in trf4Delta cells also requires Rex1p. Overexpression of the La protein Lhp1p reduces both exonucleolytic pre-tRNA trimming in wild-type cells and the accumulation of defective RNAs in trf4Delta cells. Our experiments reveal that one consequence of Rex1p-dependent 3' trimming is the generation of aberrant RNAs that are targeted for decay by TRAMP.     

Bioconversion of Cellulose to Acetate with Pure Cultures of Ruminococcus albus and a Hydrogen-Using Acetogen

Bioconversion of cellulose to acetate was accomplished with cocultures of two organisms. One was the cellulolytic species Ruminococcus albus. It ferments crystalline cellulose (Avicel) to acetate, ethanol, CO(inf2), and H(inf2). The other organism (HA) obtains energy for growth by using H(inf2) to reduce CO(inf2) to acetate. HA is a gram-negative coccobacillus that was isolated from horse feces. Coculture of R. albus with HA in batch or continuous culture alters the fermentation products formed from crystalline cellulose by the ruminococcus via interspecies H(inf2) transfer. The major product of the fermentation by R. albus and HA coculture is acetate. High concentrations of acetate (333 mM) were obtained when batch cocultures grown on 5% cellulose were neutralized with Ca(OH)(inf2). Continuous cocultures grown at retention times of 2 and 3.1 days produced 109 and 102 mM acetate, respectively, when fed 1% cellulose with utilization of 84% of the substrate.     

Bacterial noncoding Y RNAs are widespread and mimic tRNAs

Many bacteria encode an ortholog of the Ro60 autoantigen, a ring-shaped protein that is bound in animal cells to noncoding RNAs (ncRNAs) called Y RNAs. Studies in Deinococcus radiodurans revealed that Y RNA tethers Ro60 to polynucleotide phosphorylase, specializing this exoribonuclease for structured RNA degradation. Although Ro60 orthologs are present in a wide range of bacteria, Y RNAs have been detected in only two species, making it unclear whether these ncRNAs are common Ro60 partners in bacteria. In this study, we report that likely Y RNAs are encoded near Ro60 in >250 bacterial and phage species. By comparing conserved features, we discovered that at least one Y RNA in each species contains a domain resembling tRNA. We show that these RNAs contain nucleotide modifications characteristic of tRNA and are substrates for several enzymes that recognize tRNAs. Our studies confirm the importance of Y RNAs in bacterial physiology and identify a new class of ncRNAs that mimic tRNA.     

A new lamin in Xenopus somatic tissues displays strong homology to human lamin A

The nuclear lamina of vertebrates is composed of several major polypeptides that range in mol. wt from 60 to 80 kd. In mammals, the three major lamin proteins are designated A, B and C. Two major lamins have been described in Xenopus somatic tissues; two other lamins are expressed primarily in germ cells. We have analysed a cDNA clone encoding a Xenopus lamin that is highly homologous to human lamins A and C. The predicted protein has the carboxy-terminal domain characteristic of human lamin A and is thus a lamin A homologue. Surprisingly, the lamin encoded by the cDNA clone is not one of the known Xenopus lamins. The encoded protein is distinct in size from the oocyte lamin LIII and the two somatic lamins LI and LII. Monoclonal antibodies specific for LII, LIII and LIV (the lamin of male germ cells) do not recognize the protein encoded by the cDNA clone; conversely, a polyclonal antibody against the encoded protein does not recognize any of the known Xenopus lamins. This lamin is expressed late in embryonic development, and is present in all adult somatic cells examined, except erythrocytes. Thus frogs and mammals are similar in having three major somatic lamins that fall into distinct structural classes.     

Bradykinin induces superoxide anion release from human endothelial cells

The time-dependent release of superoxide anion (O2-) from bradykinin (Bk)-stimulated human umbilical vein endothelial cells (EC) was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome C employing a novel application of microspectrophotometry. In the absence of Bk, O2- release by EC was not detectable. EC exposure to Bk (10(-6) to 10(-5) M) resulted in a rapid release of O2-. The release of O2- occurred within 5 minutes of exposure. O2- release was partially inhibited by indomethacin (63 +/- 6%), thus suggesting that arachidonic acid metabolism, through cyclooxygenase, contributes to EC O2- production. EC O2- release may be an important component in the pathophysiologic actions of Bk on vascular function.