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Niyati Jain Christopher E. Morgan Brittany D. Rife Marco Salemi Blanton S. Tolbert 《The Journal of biological chemistry》2016,291(5):2331-2344
Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3′ acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein. 相似文献
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Christian Paech John Pierce Stephen D. McCurry N.E. Tolbert 《Biochemical and biophysical research communications》1978,83(3):1084-1092
Xylulose-1,5-bisphosphate in preparations of ribulose-1,5-bisphosphate (ribulose-P2) arises from non-enzymic epimerization and inhibits the enzyme. Another inhibitor, a diketo degradation product from ribulose-P2, is also present. Both compounds simulate the substrate inhibition of ribulose-P2 carboxylase/oxygenase previously reported for ribulose-P2. Freshly prepared ribulose-P2 had little inhibitory activity. The instability of ribulose-P2 may be one reason for a high level of ribulose-P2 carboxylase in chloroplasts where the molarity of active sites exceeds that of ribulose-P2. Because the KD of the enzyme/substrate complex is ≤1 μM, all ribulose-P2 generated in situ may be stored as this complex to prevent decomposition. 相似文献
5.
David B. Collinge Dawn E. Milligan J. Maxwell Dow Graham Scofield Michael J. Daniels 《Plant molecular biology》1987,8(5):405-414
Xanthomonas campestris pv. vitians, a pathogen of lettuce, elicits a hypersensitive response within 12 hours of inoculation into Brassica leaves, characterized by tissue collapse, loss of membrane integrity, vein blockage and melanin production. In contrast, the compatible pathogen, X. c. pv. campestris, has no visible effects on leaves for 48 hours, after which inoculated areas show chlorosis which eventually spreads, followed by rotting.mRNA was prepared from leaves inoculated with suspensions of both pathovars or with sterile medium up to 24 hours following inoculation. In vitro translation of total and poly A+ RNA in rabbit reticulocyte lysate in the presence of 35S methionine followed by separation of the polypeptide products by 2D-PAGE, allowed comparison of the effects of these treatments on plant gene expression. Major changes in gene expression were observed as a consequence of the inoculation technique. In addition, after inoculation with X. c. vitians, up to fifteen additional major polypeptides appeared or greatly increased by four hours. Some of these had disappeared by nine hours and several more had appeared. No major polypeptides disappeared or decreased greatly in intensity following inoculation with X. c. vitians. 相似文献
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Two isoforms of dihydroxyacetone phosphate reductase were present in Dunaliella tertiolecta. The major form was located in the chloroplast and the minor form in the cytosol. The chloroplastic reductase eluted first from a DEAE cellulose column followed immediately by the cytosolic form. Both forms were unstable and cold labile. Addition of 5 millimolar dithiothreitol helped to stabilize the enzymes. The cytosolic isoform of DHAP reductase was detected only if the cells were in an active log phase of growth. Then its activity was 20 to 30% of the total reductase activity. When cell cultures entered late log phase of growth the activity of the cytosolic form of the enzyme disappeared, but the chloroplastic form remained. The cytosolic DHAP reductase from Dunaliella has some properties similar to the cytosolic isoform from spinach leaves. Detergents inhibited both enzymes. However, neither form of the algal dihydroxyacetone phosphate reductase was stimulated by fructose 2,6-bisphosphate. In Dunaliella the properties of the chloroplastic form were those expected for glycerol production for osmoregulation, whereas the cytosolic form, like the reductases in leaves, is more likely involved in glycerol phosphate formation for lipid synthesis. 相似文献
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Pseudomonas aeruginosa outer membrane protein F produced inEscherichia coli retains vaccine efficacy
Janice M. Matthews-Greer Dawn E. Robertson Linda B. Gilleland Dr. Harry E. Gilleland Jr. 《Current microbiology》1990,20(3):171-175
Pseudomonas aeruginosa outer membrane protein F was purified by extraction from polyacrylamide gels of cell envelope proteins of anEscherichia coli strain expressing the cloned gene for protein F. Antisera directed against protein F purified fromP. aeruginosa PAO1 reacted with thisE. coli strain by immunofluorescence assay and immunoblotting, whereas these antisera were nonreactive withE. coli strains lacking thePseudomonas protein F gene. The protein F purified from thisE. coli strain was used to immunize mice by intramuscular injection of 10 µg of protein F preparation on days 1 and 14, followed by burn and challenge of the mice on day 28. As compared with control mice immunized withE. coli K-12 lipopolysaccharide, immunization with theE. coli-derived protein F afforded significant protection against subsequent challenge with heterologous Fisher-Devlin immunotype 5 and 6 strains ofP. aeruginosa. Antisera from mice immunized with theE. coli-derived protein F reacted at bands corresponding to protein F and 2-mercaptoethanol-modified protein F upon immunoblotting against cell envelope proteins of the PAO1, immunotype 5, and immunotype 6 strains ofP. aeruginosa and theE. coli strain containing the cloned F gene, but failed to react at these sites in anE. coli strain lacking the F gene. These data demonstrate thatP. aeruginosa protein F produced inE. coli through genetic engineering techniques retains its vaccine efficacy in the complete absence of anyP. aeruginosa lipopolysaccharide. 相似文献
8.
Cells of Dunaliella tertiolecta from the log phase of growth were broken by rapid extrusion at low pressure through a Yeda press and the chloroplasts were isolated by centrifugation through a Percoll gradient. Osmolarity of the growth media, the suspending media, and the Percoll gradient was kept identical to minimize change in chloroplast volume and mitochondrial entrapment. The isolated intact chloroplasts were obtained in a 30 to 50% yield based on chlorophyll and were stable to washing with buffered medium. Isolated chloroplast yield and purity was dependent on cell culture condition; a cycle of 16 hours light and 8 hours dark with continuous high CO2 was optimum. Isolated chloroplasts were about 90% intact by microscopic examination, ferricyanide-dependent O2 evolution, and the distribution of four stromal enzymes. Enzymes associated with glycolate metabolism were not in the chloroplast fraction. The isolated chloroplasts with 10 millimolar bicarbonate evolved 24 micromoles of O2 and fixed 21 micromoles of CO2 per hour per milligram of chlorophyll, which rates were about one-third of those by whole cells. The inhibition of oxygen evolution by 10 millimolar phosphate was reversed by P-glycerate. Whole chloroplasts were also isolated from cells adapted to low CO2 in air for 24 hours. On low CO2 the cells excreted more gelatinous material, which had to be removed with additional washing of the cells, before it was possible to obtain good chloroplast preparations. 相似文献
9.
James V. Moroney N. E. Tolbert Barbara B. Sears 《Molecular & general genetics : MGG》1986,204(2):199-203
Summary Six independently isolated mutants of Chlamydomonas reinhardtii that require elevated CO2 for photoautotrophic growth were tested by complementation analysis. These mutants are likely to be defective in some aspect of the algal concentrating mechanism for inorganic carbon as they exhibit CO2 fixation and inorganic carbon accumulation properties different from the wild-type. Four of the six mutants defined a single complementation group and appear to be defective in an intracellular carbonic anhydrase. The other two mutations represent two additional complementation groups.Abbreviations HS
high salt medium which has 13 mM phosphate at pH 6.8
- HSA
high salt plus 36 mM acetate medium
- YA
high salt medium with 4 g yeast extract per L and 36mM acetate
- Arg
arginine
- cia-
CO2 accumulation mutants that cannot grow on low CO2
- Ci
inorganic carbon (CO2+HCO
-
3
)
- CA
carbonic anhydrase
- mt
mating type
Supported in part by the McKnight Foundation and by NSF grant PCM 8005917 and published as journal article 11924 from the Michigan State Agriculatural Experiment Station 相似文献
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