Biomedical subjects

E G Niles

Publications and source records attributed to E G Niles.

At least 19 recordsLinked to original sources

PubMed2001

Intracellular chelation of iron by bipyridyl inhibits DNA virus replication: ribonucleotide reductase maturation as a probe of intracellular iron pools.

The efficient replication of large DNA viruses requires dNTPs supplied by a viral ribonucleotide reductase. Viral ribonucleotide reductase is an early gene product of both vaccinia and herpes simplex virus. For productive infection, the apoprotein must scavenge iron from the endogenous, labile iron pool(s). The membrane-permeant, intracellular Fe(2+) chelator, 2,2'-bipyridine (bipyridyl, BIP), is known to sequester iron from this pool. We show here that BIP strongly inhibits the replication of both vaccinia and herpes simplex virus, type 1. In a standard plaque assay, 50 microm BIP caused a 50% reduction in plaque-forming units with either virus. Strong inhibition was observed only when BIP was added within 3 h post-infection. This time dependence was observed also in regards to inhibition of viral late protein and DNA synthesis by BIP. BIP did not inhibit the activity of vaccinia ribonucleotide reductase (RR), its synthesis, nor its stability indicating that BIP blocked the activation of the apoprotein. In parallel with its inhibition of vaccinia RR activation, BIP treatment increased the RNA binding activity of the endogenous iron-response protein, IRP1, by 1.9-fold. The data indicate that the diiron prosthetic group in vaccinia RR is assembled from iron taken from the BIP-accessible, labile iron pool that is sampled also by ferritin and the iron-regulated protein found in the cytosol of mammalian cells.

2,2'-Dipyridyl↗

PubMed2001

The viral RNA polymerase H4L subunit is required for Vaccinia virus early gene transcription termination.

Vaccinia virus early gene transcription is catalyzed by a multisubunit virion form of RNA polymerase that possesses a unique subunit, H4L. Prior studies from this laboratory showed that the NH(2)-terminal domain of H4L, containing amino acids 1-195, interacts with the COOH-terminal end of nucleoside triphosphate phosphohydrolase I (NPH I), an ATPase that is employed in early gene transcription termination. Carboxyl-terminal deletion mutations of NPH I lose both the ability to mediate transcription termination and binding to H4L, providing evidence that the interaction between NPH I and H4L is required for termination. In order to test this model further, antibodies raised against segments of H4L were tested for their ability to inhibit transcription termination in vitro. A bead-bound template was employed in these studies, which permitted us to separate transcription initiation from elongation and termination. Antibodies raised against H4L amino acids 1-256 inhibited termination in an in vitro assay using virus-infected cell extracts lacking NPH I, but antibodies raised against H4L amino acids 568-795 did not. Preincubation of anti-H4L(1-256) antibodies with H4L fragments 1-256 or 1-195 prevented antibody inhibition of termination, demonstrating that inhibition was mediated by antibody binding to one or more epitopes in the NH(2)-terminal end of H4L. Antibody inhibition of termination is reduced in wild type virus-infected cell extracts containing NPH I. Furthermore, preincubation of a NPH I minus cell extract with NPH I prior to antibody addition, or readdition of NPH I to isolated ternary complexes prepared in the absence of NPH I, prevented antibody inhibition of transcription termination. These data show that NPH I and the inhibitory antibodies compete for a binding site(s) on H4L, providing further evidence that the H4L subunit of the vaccinia virus RNA polymerase plays a direct role in transcription termination.

Animals↗

PubMed2001

Interaction between the J3R subunit of vaccinia virus poly(A) polymerase and the H4L subunit of the viral RNA polymerase.

J3R, the 39-kDa subunit of vaccinia virus poly(A) polymerase, is a multifunctional protein that catalyzes (nucleoside-2'-O-)-methyltransferase activity, serves as a poly(A) polymerase stimulatory factor, and acts as a postreplicative positive transcription elongation factor. Prior results support an association between poly(A) polymerase and the virion RNA polymerase. A possible direct interaction between J3R and H4L subunit of virion RNA polymerase was evaluated. J3R was shown to specifically bind to H4L amino acids 235-256, C terminal to NPH I binding site on H4L. H4L binds to the C-terminal region of J3R between amino acids 169 and 333. The presence of a J3R binding site near to the NPH I binding region on H4L led us to evaluate a physical interaction between NPH I and J3R. The NPH I binding site was located on J3R between amino acids 169 and 249, and J3R was shown to bind to NPH I between amino acids 457 and 524. To evaluate a role for J3R in early gene mRNA synthesis, transcription termination, and/or release, a transcription-competent extract prepared from cells infected with mutant virus lacking J3R, J3-7. Analysis of transcription activity demonstrated that J3R is not required for early mRNA synthesis and is not an essential factor in early gene transcription termination or transcript release in vitro. J3R interaction with NPH I and H4L may serve as a docking site for J3R on the virion RNA polymerase, linking transcription to mRNA cap formation and poly(A) addition.

Animals↗

PubMed2001

Identification and characterization of a Smad2 homologue from Schistosoma mansoni, a transforming growth factor-beta signal transducer.

Smad proteins are essential intracellular signal transducers of the transforming growth factor-beta (TGF-beta) superfamily. The TGF-beta superfamily signals through phosphorylation and activation of R-Smad proteins, receptor-regulated Smads, by heteromeric complexes of ligand-specific type I and type II serine/threonine kinase receptors. R-Smads receive a signal from the activated receptor complex and transmit it to the nucleus. A cDNA was isolated that encodes a 649-amino acid protein found to be homologous to members of R-Smad subfamily with highest homology scored to clawed African frog and human Smad2. The Schistosoma mansoni homologue (SmSmad2) was overexpressed in bacteria as a Sj26-GST fusion protein and used to raise specific antibodies. The IgG fraction of the immunized rabbit serum identified 70- and 72-kDa protein bands in Western analysis of schistosome extracts. Treatment with alkaline phosphatase removed the 72-kDa band, which indicates that this band represents the phosphorylated form of schistosome Smad2. SmSmad2 was localized in the subtegument, parenchymal cells, and sex organs in both male and female worm cryosections. Similar results were also obtained from the analysis of the Smad2 mRNA distribution pattern revealed by in situ hybridization of adult worm pair paraffin sections. SmSmad2 mRNA levels were determined by reverse transcriptase-polymerase chain reaction in different mammalian host developmental stages and found to be constitutively expressed. SmSmad2 was also found to interact with a previously identified SmTbetaR-I, a serine/threonine type I kinase receptor. Furthermore, SmSmad2 was shown to undergo phosphorylation by constitutively active forms of SmTbetaR-I in vitro. In addition, SmSmad2 localized in the nuclei of mink lung epithelial cells upon treatment with TGF-beta(1). These data indicate that the SmSmad2 responds to the TGF-beta signals by interaction with receptor I, which phosphorylates it, whereupon it translocates into the nucleus presumably to regulate target gene transcription and consequently elicit a specific TGF-beta effect.

Amino Acid Sequence↗

PubMed2001

Evaluation of Schistosoma mansoni retinoid X receptor (SmRXR1 and SmRXR2) activity and tissue distribution.

Recently, we reported the identification of cDNA's encoding retinoid X receptor (RXR) homologues in Schistosoma mansoni. RXRs are known to be involved in the regulation of genes important for homeostasis and development. Previous studies indicated that SmRXR1 plays a role in the regulation of the female-specific gene, p14. Herein, we report that SmRXR2 also binds to cis-elements present in the p14 upstream region when evaluated in yeast reporter strains. SmRXR2 shows a pattern of recognition of cis-sequences present in the p14 gene upstream region different than SmRXR1. However, the SmRXR2 C (DNA binding) domain binds promiscuously in electrophoretic mobility shift assays to cis-elements of the p14 upstream region. The SmRXRs differ in their ability to activate transcription. The N-terminal A/B domain of SmRXR1 is necessary and sufficient for autonomous transcription activation function (AF) in yeast. SmRXR2 does not exhibit an equivalent autonomous AF. SmRXR1 and SmRXR2 fail to dimerize when investigated both in the yeast two-hybrid system and in immunoprecipitation experiments. In situ hybridization experiments using paraffin sections of adult worms demonstrate that SmRXR1 and SmRXR2 exhibit both common and unique cell type distribution which indicates that SmRXR1 and SmRXR2 both play a role in regulating gene expression in certain cells, yet each plays a distinct role in modulating the expression of genes in other cell types. Both SmRXR1 and SmRXR2 localize to vitelline cells. These studies provide a solid basis for improving our understanding of RXRs and their importance in female-specific gene regulation.

Animals↗

PubMed2000

Interaction between nucleoside triphosphate phosphohydrolase I and the H4L subunit of the viral RNA polymerase is required for vaccinia virus early gene transcript release.

Signal-dependent termination is restricted to early poxvirus genes whose transcription is catalyzed by the virion form of RNA polymerase. Two termination factors have been identified. Vaccinia termination factor/capping enzyme is a multifunctional heterodimer that also catalyzes the first three steps of mRNA cap formation and is an essential intermediate gene transcription initiation factor. Nucleoside triphosphate phosphohydrolase I (NPH I) is a single-stranded DNA-dependent ATPase. COOH-terminal deletion mutations of NPH I retain both ATPase and DNA binding activities but are unable either to terminate transcription or to act as dominant negative mutants in vitro. One appealing model posits that the COOH-terminal region of NPH I binds to one or more components in the termination complex. In an attempt to identify NPH I-related protein/protein interactions involved in transcription termination, a series of pull-down experiments were done. Among several vaccinia virus proteins tested, the H4L subunit, unique to the virion form of RNA polymerase, was shown to bind glutathione S-transferase (GST)-NPH I. To further confirm this interaction in virus-infected cells, we constructed recombinant vaccinia virus, vNPHINGST, that expresses GST-tagged NPH I. The H4L subunit of virion RNA polymerase specifically co-purified with GST-NPH I, consistent with a physical interaction. Through the analysis of a series of NH(2)- and COOH-terminal truncation mutations of H4L, the NPH I interaction site was localized to the NH(2)-terminal 195 amino acids of the H4L protein. The H4L binding site on NPH I was mapped to the COOH-terminal region between 457 and 631. Furthermore, COOH-terminal deletion mutations of NPH I failed to bind the NH(2)-terminal region of H4L, explaining their inability to support transcription termination. The COOH-terminal end of NPH I was also shown to be required for transcript release activity and for dominant negative inhibition of release. The requirement for an essential interaction between NPH I and H4L provides an explanation for the observed restriction of transcription termination to early viral genes.

Acid Anhydride Hydrolases↗

PubMed2000

Transcription elongation activity of the vaccinia virus J3 protein in vivo is independent of poly(A) polymerase stimulation.

Prior genetic analysis suggests that the vaccinia virus J3 gene product, previously characterized as a bifunctional (nucleoside-2'-O-)-methyltransferase and poly(A) polymerase stimulatory factor, is a postreplicative positive transcription elongation factor. To test this hypothesis, viruses bearing mutations in the J3 gene were characterized with respect to viral protein and RNA synthesis in infected cells. The analysis reveals that compared to wt virus infections, J3 mutants synthesize reduced amounts of large late viral proteins and shorter-than-normal intermediate and late mRNAs. Structural analysis of one late mRNA shows that it is specifically truncated from the 3' end, thus accounting for its shorter than normal chain length. Thus J3 mutant viruses are defective in elongation of transcription of postreplicative viral genes, strongly suggesting that the J3 gene product normally acts as a positive transcription elongation factor. Biochemical analysis of one J3 missense mutant demonstrates that it retains poly(A) stimulatory activity but is defective in (nucleoside-2'-O-)-methyltransferase activity. Thus the elongation factor activity of the J3 gene product is independent of the poly(A) stimulatory activity. It remains to be determined whether the (nucleoside-2'-O-)-methyltransferase and elongation factor activities of the J3 protein are linked or can be uncoupled by mutation.

Animals↗

PubMed2000

Identification and functional characterization of a member of the PUR-alpha family from Schistosoma mansoni.

Schistosoma mansoni p14 gene encodes an eggshell precursor that is expressed only in vitelline cells of mature female worms in response to a male stimulus. The upstream region of the p14 gene contains several potential cis-acting regulatory sequences. We used the upstream region of the p14 gene as bait in a yeast-one-hybrid screen of a S. mansoni cDNA library to identify interacting proteins. We report the identification and characterization of a cDNA (S. mansoni PUR-alpha (SmPUR-alpha)) encoding a protein homologous to single-stranded DNA transcription activator PUR-alpha, that binds to the p14 upstream region and activates transcription of the HIS3 reporter gene in yeast. SmPUR-alpha has a predicted molecular mass of 30 kDa and shares an overall homology of 63% with mammalian PUR-alpha. The DNA binding domain of SmPUR-alpha is highly conserved. We show by gel shift assays that GST-SmPUR-alpha binds to oligonucleotides comprising the p14 upstream region. SmPUR-alpha binds preferentially to single-stranded DNA and also binds RNA. Unlike the mammalian homologue, SmPUR-alpha exhibits little specificity for the PUR element GGn, but shows strong preference for a sequence containing alternating pyrimidines. Our data support that SmPUR-alpha is a single-copy gene and through reverse transcriptase-polymerase chain reaction and in situ hybridization, we show that SmPUR-alpha is constitutively transcribed in many cell types and thus likely plays a role as a general transcription activator in schistosomes.

Amino Acid Sequence↗

PubMed1999

E2/E3-mediated assembly of lysine 29-linked polyubiquitin chains.

Polyubiquitin (Ub) chains linked through Lys-48-Gly-76 isopeptide bonds represent the principal signal by which substrates of the Ub-dependent protein degradation pathway are targeted to the 26 S proteasome, but the mechanism(s) whereby these chains are assembled on substrate proteins is poorly understood. Nor have assembly mechanisms or definitive functions been assigned to polyubiquitin chains linked through several other lysine residues of ubiquitin. We show that rabbit reticulocyte lysate harbors enzymatic components that catalyze the assembly of unanchored Lys-29-linked polyubiquitin chains. This reaction can be reconstituted using the ubiquitin-conjugating enzyme (E2) known as UbcH5A, a 120-kDa protein(s) that behaves as a ubiquitin-protein ligase (E3), and ubiquitin-activating enzyme (E1). The same partially purified E3 preparation also catalyzes the assembly of unanchored chains linked through Lys-48. Kinetic studies revealed a K(m) of approximately 9 microM for the acceptor ubiquitin in the synthesis of diubiquitin; this value is similar to the concentration of free ubiquitin in most cells. Similar kinetic behavior was observed for conjugation to Lys-48 versus Lys-29 and for conjugation to tetraubiquitin versus monoubiquitin. The properties of these enzymes suggest that there may be distinct pathways for ubiquitin-ubiquitin ligation versus substrate-ubiquitin ligation in vivo.

Animals↗

PubMed1999

Interaction of the vaccinia virus nucleoside triphosphate phosphohydrolase I with linear oligonucleotides.

Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH I) serves as the ATPase activity employed in early gene transcription termination [Deng, L., and Shuman, S. (1998) Genes Dev. 12, 538-546; Christen, L. M., et al. (1998) Virology 245, 360-371]. Since ATPase activity requires binding of single-stranded DNA, a full understanding of the mechanism of oligonucleotide activation is essential for the elucidation of its role in transcription termination. To initiate detailed structure-function studies of NPH I, we undertook combined kinetic and binding analyses of the interaction of linear oligonucleotides with NPH I. In the presence of single-stranded DNA, ATP exhibits complex saturation kinetics. The apparent Km for ATP is independent of DNA concentration, demonstrating that ssDNA binding alters the kcat for the reaction. Linear ssDNA oligonucleotides from 18 to 48 nucleotides in length stimulated activity in a saturatable fashion. As the oligonucleotide length increases, the Kact decreases and the Vmax increases. The increase in affinity is paralleled by an increase in the level of binding as measured by EMSA. The kinetic activation observed for 36-nucleotide ssDNA is dependent upon ATP concentration. At low ATP levels, sigmoidal saturation kinetics are observed, while at saturating ATP levels, near-hyperbolic kinetics are seen, suggesting that NPH I may adopt two conformational states. Linear oligonucleotides 18, 24, and 36 bases in length bind one, two, and three molecules of NPH I maximally, respectively, indicating that the NPH I binding site is no more than 12 bases in length. In contrast, single-stranded RNA does not stimulate ATPase activity, yet RNA binds as well as DNA of a similar length. Both RNA and DNA can be photo-cross-linked to NPH I by UV light. ssDNA and ssRNA cross-compete in UV photo-cross-linking to NPH I, indicating that both oligonucleotides share a common binding site. ssRNA prevents ssDNA activation of ATPase activity, confirming that both oligonucleotides bind to the kinetically important oligonucleotide activation site on NPH I. ssDNA inhibits transcription termination in vitro. Inhibition is overcome by adding NPH I, demonstrating that oligonucleotide inhibition is mediated through NPH I.

Acid Anhydride Hydrolases↗

PubMed1999

RXR-2, a member of the retinoid x receptor family in Schistosoma mansoni.

A cDNA encoding a second full-length member of the Schistosoma mansoni RXR family (SmRXR-2) was identified. The nucleotide sequence of SmRXR-2 translates into a protein of 784 amino acids with a pI of 7.63 and an approximate mass of 78kDa making it the largest reported RXR to date. Phylogenetic tree analysis provides evidence that SmRXR-2 is the most ancient full-length RXR identified. SmRXR-2 exhibits unique sequence features compared with other RXRs. RT-PCR results demonstrate that the SmRXR-2 gene is constitutively expressed and thus must play multiple roles throughout schistosome development in the vertebrate host.

Amino Acid Sequence↗

PubMed1999

Characterization of the Ras homologue of Schistosoma mansoni.

Ras is a member of a super-family of guanine-binding or G-proteins. Ras functions as a molecular switch in the transduction of signals generated by the activation of a variety of cell surface receptors and relays the signals to downstream effectors. Little is known about signal transduction in schistosomes. In order for Schistosoma mansoni to survive different immune responses triggered by the host as well as to migrate from the site of penetration at the skin to the final destination in portal circulation, they must receive signals from the host environment and respond to them in a way that allows their survival. We have isolated the schistosome Ras cDNA by using sequence information of the schistosome Ras homologue submitted to the Genbank database. Analysis of the encoded peptide revealed 81% identity and 92% similarity with K-Ras from various species. Ras is a single copy gene as determined by quantitative hybridization experiments. The cDNA was cloned into pGEX-4T and the expressed peptide was used to generate specific antibody reagents. Affinity purified antibodies identified a 23 kDa native protein that localizes to the subtegument. Ras is not associated with the tegument. Ras is expressed in all the developmental stages of the parasite. However, Ras is over-expressed in female worms compared to males. Schistosome Ras was also shown to be post-translationally modified by addition of farnesyl isoprenoid moiety to the cysteine residue in the C-terminal box. Using a schistosome extract in vitro SmRas farnesylation was inhibited by the farnesyl transferase inhibitor, FTI-277, at concentrations comparable to those required to inhibit K-Ras processing. These initial studies on signal transduction in schistosomes should provide a solid basis for improving our understanding of schistosome-host interactions.

Alkyl and Aryl Transferases↗

PubMed1999

Identification of a cDNA encoding a retinoid X receptor homologue from Schistosoma mansoni. Evidence for a role in female-specific gene expression.

Schistosoma mansoni, a multicelluar eukaryotic blood fluke, is a major cause of morbidity worldwide in humans. The study of female parasite growth, development, and gene regulation is important because the eggs produced are responsible for the pathogenesis observed in schistosomiasis. p14, an eggshell precursor gene expressed only in sexually mature females in response to a male stimulus, is a model for female-specific gene regulation. The upstream region of the p14 gene shares sequences present in insect genes known to be regulated in a sex-, temporal-, and tissue-specific manner by members of the steroid receptor superfamily. Herein, we report the identification and characterization of a cDNA that encodes the S. mansoni (Sm) RXR homologue. Sequence analysis predicts and Western blot analysis confirms the synthesis of a 74-kDa protein, the largest member of the RXR family reported to date. We show by electrophoretic mobility shift assay analysis that SmRXR binds to cis-elements of the p14 gene including a direct repeat that follows the "3-4-5" rule of binding elements recognized by members of the steroid receptor superfamily. Furthermore, we demonstrate that SmRXR can act as a transcription activator in the yeast one-hybrid system. Through quantitative reverse transcriptase-polymerase chain reaction, we show that the SmRXR gene is constitutively expressed and thus must play multiple roles throughout the schistosome life cycle.

Amino Acid Sequence↗

PubMed1998

Core domain mutation (S86Y) selectively inactivates polyubiquitin chain synthesis catalyzed by E2-25K.

The mammalian ubiquitin conjugating enzyme known as E2-25K catalyzes the synthesis of polyubiquitin chains linked exclusively through K48-G76 isopeptide bonds. The properties of truncated and chimeric forms of E2-25K suggest that the polyubiquitin chain synthesis activity of this E2 depends on specific interactions between its conserved 150-residue core domain and its unique 50-residue tail domain [Haldeman, M. T., Xia, G., Kasperek, E. M., and Pickart, C. M. (1997) Biochemistry 36, 10526-10537]. In the present study, we provide strong support for this model by showing that a point mutation in the core domain (S86Y) mimics the effect of deleting the entire tail domain: the ability to form an E2 approximately ubiquitin thiol ester is intact, while conjugation activity is severely inhibited (>/=100-fold reduction in kcat/Km). The properties of E2-25K enzymes carrying the S86Y mutation indicate that this mutation strengthens the interaction between the core and tail domains: both free and ubiquitin-bound forms of S86Y-25K are completely resistant to tryptic cleavage at K164 in the tail domain, whereas wild-type enzyme is rapidly cleaved at this site. Other properties of S86Y-26K suggest that the active site of this mutant enzyme is more occluded than the active site of the wild-type enzyme. (1) Free S86Y-25K is alkylated by iodoacetamide 2-fold more slowly than the wild-type enzyme. (2) In assays of E2 approximately ubiquitin thiol ester formation, S86Y-25K shows a 4-fold reduced affinity for E1. (3) The ubiquitin thiol ester adduct of S86Y-25K undergoes (uncatalyzed) reaction with dithiothreitol 3-fold more slowly than the wild-type thiol ester adduct. One model to accommodate these findings postulates that an enhanced interaction between the core and tail domains, induced by the S86Y mutation, causes a steric blockade at the active site which prevents access of the incoming ubiquitin acceptor to the thiol ester bond. Consistent with this model, the S86Y mutation inhibits ubiquitin transfer to macromolecular acceptors (ubiquitin and polylysine) more strongly than transfer to small-molecule acceptors (free lysine and short peptides). These results suggest that unique residues proximal to E2 active sites may influence specific function by mediating intramolecular interactions.

Alkylation↗

PubMed1998

Vaccinia virus nucleoside triphosphate phosphohydrolase I is an essential viral early gene transcription termination factor.

Deng and Shuman (J. Biol Chem. 271, 29386 (1996)) reported that an ATPase different from the known viral termination factor, VTF, is required for vaccinia virus early gene transcription termination. Properties of this ATPase were similar to those of a known vaccinia virus enzyme, nucleoside triphosphate phosphohydrolase I (NPH I) the product of gene D11L. Transcription-competent cell-free extracts were prepared from A549 cells infected with wild-type or mutant vaccinia virus harboring ts mutations in gene D11L. These extracts were employed to investigate the role of NPH I in early gene transcription termination. Extracts prepared under nonpermissive conditions from both wild-type virus and ts mutant virus-infected cells exhibited high levels of early and intermediate gene transcription activity but were incapable of supporting late gene transcription. ts mutant extract lacked signal-dependent early gene transcription termination activity, which was restored by the addition of either free NPH I or a GST-NPH I fusion protein. A comparison of the NPH I amino acid sequence to the protein databases revealed the presence of a set of sequences characteristic of nucleic acid helicase superfamily II members. A series of site-specific mutations in the helicase motifs and N-terminal and C-terminal deletion mutations were expressed as GST fusion proteins and their activities assessed. Of the mutations in helicase motifs I to VI, alteration of all but motif III reduced the ATPase activity. Removal of as few as 24 amino acids from the N-terminal end eliminated ATPase activity, while deletion of 68 C-terminal amino acids exhibited only a modest decrease in ATP hydrolysis. Larger C-terminal deletions eliminated ATPase activity. Each deletion mutation, and site-specific mutations other than the motif III mutation, failed to support transcription termination in vitro. Mutations in motifs I, II, V, and VI inhibit wild-type NPH I transcription termination activity. However, deletion of up to 68 amino acids from the C-terminal end eliminates this inhibitory property. This observation is particularly interesting since these C-terminal deletions retain both ATPase activity and single-stranded DNA binding activity. Their failure to inhibit transcription termination suggests that these C-terminal deletion mutations eliminate a site required for a function other than from DNA binding or ATP hydrolysis.

Acid Anhydride Hydrolases↗

PubMed1996

Domain structure of the vaccinia virus mRNA capping enzyme. Expression in Escherichia coli of a subdomain possessing the RNA 5'-triphosphatase and guanylyltransferase activities and a kinetic comparison to the full-size enzyme.

The RNA 5'-triphosphatase, nucleoside triphosphate phosphohydrolase, and guanylyltransferase activities of the vaccinia virus mRNA capping enzyme were previously localized to an NH2-terminal 60-kDa domain of the D1R subunit. Measurement of the relative ATPase and guanylyltransferase activities remaining in D1R carboxyl-terminal deletion variants expressed in Escherichia coli BL21(DE3)plysS localizes the carboxyl terminus of the active domain to between amino acids 520 and 545. Failure to obtain a deletion mutant with the loss of one activity indicates that the catalysis of both reactions requires a common domain structure. Based on these results, a truncated D1R protein terminating at amino acid 545 was expressed in E. coli and purified to homogeneity. D1R1-545 was found to be kinetically equivalent to the holoenzyme in regard to ATPase, RNA 5'-triphosphatase, and guanylyltransferase activities. Measurement of RNA binding by mobility shift and UV photo-cross-linking analyses also demonstrates the ability of this domain to bind RNA independent of the methyltransferase domain, comprised of the carboxyl terminus of D1R from amino acids 498-844 and the entire D12L subunit. RNA binding to D1R1-545 is substantially weaker than binding to either the methyltransferase domain or the holoenzyme. Binding is inhibited by 5'-OH RNA and to a lesser extent by DNA oligonucleotides in a concentration dependent manner which correlates with the inhibition of RNA 5'-triphosphatase activity by these same oligonucleotides. We conclude that D1R1-545 represents a functionally independent domain of the mRNA capping enzyme, fully competent in substrate binding and catalysis at both the triphosphatase and guanylyltransferase active sites.

Acid Anhydride Hydrolases↗

PubMed1996

Characterization of the vaccinia virus RNA 5'-triphosphatase and nucleotide triphosphate phosphohydrolase activities. Demonstrate that both activities are carried out at the same active site.

D1R1-545, an active subdomain of the large subunit of vaccinia virus mRNA capping enzyme possessing ATPase, RNA 5'-triphosphatase, and guanylyltransferase activities, was expressed in Escherichia coli and shown to be functionally equivalent to the heterodimeric enzyme (Myette, J. R., and Niles, E. G. (1996) J. Biol. Chem. 271, 11936-11944). A detailed characterization of the phosphohydrolytic activities of D1R1-545 demonstrates that, in addition to ATPase and RNA 5'-triphosphatase activities, the capping enzyme also possesses a general nucleoside triphosphate phosphohydrolase activity that lacks a preference for the nucleoside base or sugar. Nucleoside triphosphate and mRNA saturation kinetics are markedly different, with RNA exhibiting a Km and turnover number 100- and 10-fold less, respectively, than those values measured for any NTP. The linear competitive inhibition of RNA 5'-triphosphatase activity by ATP, and the relative manner by which both ATPase and RNA 5'-triphosphatase activities are inhibited by specific oligonucleotides, kinetically demonstrate that each activity is carried out at a common active site. Direct UV photo-cross-linking of either 32P-radiolabeled ATP or 23-mer triphosphorylated RNA, followed by cyanogen bromide cleavage of the photo-linked enzyme, localizes the major binding site for both ATP and RNA to a region between amino acids 1 and 221. The inability of ATP to competitively inhibit either E approximately GMP formation or the transfer of GMP to RNA kinetically differentiates the phosphohydrolase active site from the guanylyltransferase active site.

Acid Anhydride Hydrolases↗

PubMed1996

Use of lysolecithin-permeabilized infected-cell extracts to investigate the in vitro biochemical phenotypes of poxvirus ts mutations altered in viral transcription activity.

Lysolecithin permeabilization of vaccinia virus-infected cells was employed to prepare extracts that support faithful transcription initiation in vitro on plasmids possessing early, intermediate, and late viral gene promoters. Conditions which optimize transcription from each promoter were defined. The in vitro system was used to investigate the multifunctional viral mRNA capping enzyme, which also functions as the viral early gene transcription termination factor (VTF) and a viral intermediate gene transcription initiation factor. A low level of signal-dependent termination of early gene transcription was observed in vitro which could be elevated by the addition of pure mRNA capping enzyme. VTF-dependent transcription termination was found to be restricted to templates that possessed an early promoter. This restriction mimics that observed in vivo and demonstrates that transcription termination is limited to RNA polymerase molecules that recognize early rather than intermediate or late gene promoters. Extracts prepared from cells infected at the nonpermissive temperature with a virus containing a ts mutation in gene D12L, which encodes the small subunit of VTF, are incapable of supporting both early gene transcription termination and intermediate gene transcription initiation. Both activities are restored upon addition of the purified wild-type mRNA capping enzyme.

Base Sequence↗