Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I

Cleavage and polyadenylation of mRNA 3′ ends in Saccharomyces cerevisiae requires several factors, one of which is cleavage factor I (CF I). Purification of CF I activity from yeast extract has implicated numerous proteins as functioning in both cleavage and/or polyadenylation. Through reconstitution of active CF I from separately expressed and purified proteins, we show that CF I contains five subunits, Rna14, Rna15, Pcf11, Clp1, and Hrp1. These five are necessary and sufficient for reconstitution of cleavage activity in vitro when mixed with CF II, and for specific polyadenylation when mixed with polyadenylation factor I, purified poly(A) polymerase, and poly(A) binding protein. Analysis of the individual protein–protein interactions supports an architectural model for CF I in which Pcf11 simultaneously interacts with Rna14, Rna15, and Clp1, whereas Rna14 bridges Rna15 and Hrp1.

CD95/Fas induces cleavage of the GrpL/Gads adaptor and desensitization of antigen receptor signaling

The balance between cell survival and cell death is critical for normal lymphoid development. This balance is maintained by signals through lymphocyte antigen receptors and death receptors such as CD95/Fas. In some cells, ligating the B cell antigen receptor can protect the cell from apoptosis induced by CD95. Here we report that ligation of CD95 inhibits antigen receptor-mediated signaling. Pretreating CD40-stimulated tonsillar B cells with anti-CD95 abolished B cell antigen receptor-mediated calcium mobilization. Furthermore, CD95 ligation led to the caspase-dependent inhibition of antigen receptor-induced calcium mobilization and to the activation of mitogen-activated protein kinase pathways in B and T cell lines. A target of CD95-mediated caspase 3-like activity early in the apoptotic process is the adaptor protein GrpL/Gads. GrpL constitutively interacts with SLP-76 via its C-terminal SH3 domain to regulate transcription factors such as NF-AT. Cleavage of GrpL removes the C-terminal SH3 domain so that it is no longer capable of recruiting SLP-76 to the membrane. Transfection of a truncated form of GrpL into Jurkat T cells blocked T cell antigen receptor-induced activation of NF-AT. These results suggest that CD95 signaling can desensitize antigen receptors, in part via cleavage of the GrpL adaptor.

Floral Scent Production in Clarkia breweri1 : III. Enzymatic Synthesis and Emission of Benzenoid Esters

The fragrance of Clarkia breweri (Onagraceae), a California annual plant, includes three benzenoid esters: benzylacetate, benzylbenzoate, and methylsalicylate. Here we report that petal tissue was responsible for the benzylacetate and methylsalicylate emission, whereas the pistil was the main source of benzylbenzoate. The activities of two novel enzymes, acetyl-coenzyme A:benzylalcohol acetyltransferase (BEAT), which catalyzes the acetyl esterification of benzylalcohol, and S-adenosyl-l-methionine:salicylic acid carboxyl methyltransferase, which catalyzes the methyl esterification of salicylic acid, were also highest in petal tissue and absent in leaves. In addition, the activity of both enzymes in the various floral organs was developmentally and differentially regulated. S-Adenosyl-l-methionine:salicylic acid carboxyl methyltransferase activity in petals peaked in mature buds and declined during the next few days after anthesis, and it showed a strong, positive correlation with the emission of methylsalicylate. The levels of BEAT activity and benzylacetate emission in petals also increased in parallel as the buds matured and the flowers opened, but as emission began to decline on the 2nd d after anthesis, BEAT activity continued to increase and remained high until the end of the lifespan of the flower.

Molecular and Enzymatic Characterization of Three Phosphoinositide-Specific Phospholipase C Isoforms from Potato1

Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-tris-phosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 μm Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner.

Identification and Partial Characterization of the Pectin Methyltransferase “Homogalacturonan-Methyltransferase” from Membranes of Tobacco Cell Suspensions1

A membrane preparation from tobacco (Nicotiana tabacum L.) cells contains at least one enzyme that is capable of transferring the methyl group from S-adenosyl-methionine (SAM) to the C6 carboxyl of homogalacturonan present in the membranes. This enzyme is named homogalacturonan-methyltransferase (HGA-MT) to distinguish it from methyltransferases that catalyze methyletherification of the pectic polysaccharides rhamnogalacturonan I or rhamnogalacturonan II. A trichloroacetic acid precipitation assay was used to measure HGA-MT activity, because published procedures to recover pectic polysaccharides via ethanol or chloroform:methanol precipitation lead to high and variable background radioactivity in the product pellet. Attempts to reduce the incorporation of the 14C-methyl group from SAM into pectin by the addition of the alternative methyl donor 5-methyltetrahydrofolate were unsuccessful, supporting the role of SAM as the authentic methyl donor for HGA-MT. The pH optimum for HGA-MT in membranes was 7.8, the apparent Michaelis constant for SAM was 38 μm, and the maximum initial velocity was 0.81 pkat mg−1 protein. At least 59% of the radiolabeled product was judged to be methylesterified homogalacturonan, based on the release of radioactivity from the product after a mild base treatment and via enzymatic hydrolysis by a purified pectin methylesterase. The released radioactivity eluted with a retention time identical to that of methanol upon fractionation over an organic acid column. Cleavage of the radiolabeled product by endopolygalacturonase into fragments that migrated as small oligomers of HGA during thin-layer chromatography, and the fact that HGA-MT activity in the membranes is stimulated by uridine 5′-diphosphate galacturonic acid, a substrate for HGA synthesis, confirms that the bulk of the product recovered from tobacco membranes incubated with SAM is methylesterified HGA.

Hydroperoxide lyase depletion in transgenic potato plants leads to an increase in aphid performance

Hydroperoxide lyases (HPLs) catalyze the cleavage of fatty acid hydroperoxides to aldehydes and oxoacids. These volatile aldehydes play a major role in forming the aroma of many plant fruits and flowers. In addition, they have antimicrobial activity in vitro and thus are thought to be involved in the plant defense response against pest and pathogen attack. An HPL activity present in potato leaves has been characterized and shown to cleave specifically 13-hydroperoxides of both linoleic and linolenic acids to yield hexanal and 3-hexenal, respectively, and 12-oxo-dodecenoic acid. A cDNA encoding this HPL has been isolated and used to monitor gene expression in healthy and mechanically damaged potato plants. HPL gene expression is subject to developmental control, being high in young leaves and attenuated in older ones, and it is induced weakly by wounding. HPL enzymatic activity, nevertheless, remains constant in leaves of different ages and also after wounding, suggesting that posttranscriptional mechanisms may regulate its activity levels. Antisense-mediated HPL depletion in transgenic potato plants has identified this enzyme as a major route of 13-fatty acid hydroperoxide degradation in the leaves. Although these transgenic plants have highly reduced levels of both hexanal and 3-hexenal, they show no phenotypic differences compared with wild-type ones, particularly in regard to the expression of wound-induced genes. However, aphids feeding on the HPL-depleted plants display approximately a two-fold increase in fecundity above those feeding on nontransformed plants, consistent with the hypothesis that HPL-derived products have a negative impact on aphid performance. Thus, HPL-catalyzed production of C6 aldehydes may be a key step of a built-in resistance mechanism of plants against some sucking insect pests.

Cleavage of poly(A) tails on the 3′-end of RNA by ribonuclease E of Escherichia coli

RNase E initiates the decay of Escherichia coli RNAs by cutting them internally near their 5′-end and is a component of the RNA degradosome complex, which also contains the 3′-exonuclease PNPase. Recently, RNase E has been shown to be able to remove poly(A) tails by what has been described as an exonucleolytic process that can be blocked by the presence of a phosphate group on the 3′-end of the RNA. We show here, however, that poly(A) tail removal by RNase E is in fact an endonucleolytic process that is regulated by the phosphorylation status at the 5′- but not the 3′-end of RNA. The rate of poly(A) tail removal by RNase E was found to be 30-fold greater when the 5′-terminus of RNA substrates was converted from a triphosphate to monophosphate group. This finding prompted us to re-analyse the contributions of the ribonucleolytic activities within the degradosome to 3′ attack since previous studies had only used substrates that had a triphosphate group on their 5′-end. Our results indicate that RNase E associated with the degradosome may contribute to the removal of poly(A) tails from 5′-monophosphorylated RNAs, but this is only likely to be significant should their attack by PNPase be blocked.

Energy metabolism, enzymatic flux capacities, and metabolic flux rates in flying honeybees.

Honeybees rely primarily on the oxidation of hexose sugars to provide the energy required for flight. Measurement of VCO2 (equal to VO2, because VCO2/VO2 = 1.0 during carbohydrate oxidation) during flight allowed estimation of steady-state flux rates through pathways of flight muscle energy metabolism. Comparison of Vmax values for flight muscle hexokinase, phosphofructokinase, citrate synthase, and cytochrome c oxidase with rates of carbon and O2 flux during flight reveal that these enzymes operate closer to Vmax in the flight muscles of flying honeybees than in other muscles previously studied. Possible mechanistic and evolutionary implications of these findings are discussed.

Silver ion high pressure liquid chromatography provides unprecedented separation of sterols: application to the enzymatic formation of cholesta-5,8-dien-3 beta-ol.

We report that silver ion HPLC provides remarkable separations of C27 sterols differing only in the number or location of olefinic double bonds. This technique has been extended to LC-MS, analysis of purified components by GC, GC-MS, and 1H NMR, and to its use on a semipreparative scale. The application of this methodology for the demonstration of the catalysis, by rat liver microsomes, of the conversion of 7-dehydrocholesterol to cholesta-5,8-dien-3 beta-ol is also presented.

Structure of the replication terminus-terminator protein complex as probed by affinity cleavage.

The replication terminator protein (RTP) of Bacillus subtilis is a homodimer that binds to each replication terminus and impedes replication fork movement in only one orientation with respect to the replication origin. The three-dimensional structure of the RTP-DNA complex needs to be determined to understand how structurally symmetrical dimers of RTP generate functional asymmetry. The functional unit of each replication terminus of Bacillus subtilis consists of four turns of DNA complexed with two interacting dimers of RTP. Although the crystal structure of the RTP apoprotein dimer has been determined at 2.6-A resolution, the functional unit of the terminus is probably too large and too flexible to lend itself to cocrystallization. We have therefore used an alternative strategy to delineate the three dimensional structure of the RTP-DNA complex by converting the protein into a site-directed chemical nuclease. From the pattern of base-specific cleavage of the terminus DNA by the chemical nuclease, we have mapped the amino acid to base contacts. Using these contacts as distance constraints, with the crystal structure of RTP, we have constructed a model of the DNA-protein complex. The biological implications of the model have been discussed.