Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phage SP RNA-dependent RNA polymerase (SP replicase) was purified from Escherichia coli infected with RNA phage SP. The enzyme was found to be composed of four non-identical polypeptides, i.e. subunits I, II, III, and IV and molecular weights of 74,000, 69,000, 47,000, and 36,000 daltons, respectively. As in the case of phage Qbeta replicase, the largest polypeptide is identical with the ribosomal protein S1, and subunits III and IV with polypeptide chain elongation factors EF-Tu and EF-ts, respectively.. This is based on the behaviour of the subunits on SDS-polyacrylamide gel electrophoresis, isoelectric focusing and immunological cross-reaction. Subunits I, III, and IV of SP replicase are derived from the host cell, while subunit II is coded by phage RNA genome. The striking coincidence of the composition and entity of the structural components of SP replicase with those of Qbeta replicase may indicate the structural and functional requirements of host-derived polypeptides in RNA replicase. The binding activity of S1 (in 70S ribosome comples) to poly (U) is retained in SP replicase complex. In contrast, the GDP binding activity of EF-Tu is masked in SP replicase. It is concluded that S1 is required functionally whereas EF-Tu.EF-Ts are required structurally in RNA replicase.
...
PMID:Identification of host-derived subunits of phage SP RNA-dependent RNA polymerase (SP replicase). 36 4

Escherichia coli phage Qbeta RNA replicase, an RNA-dependent RNA polymerase (RNA-dependent RNA nucleotidyltransferase), is a tetramer composed of one phage-coded polypeptide and three host-supplied polypeptides which are known to function in the biosynthesis of proteins in the uninfected host. Two of these polypeptides, protein synthesis elongation factors EF-Tu and EF-Ts, can be covalently crosslinked with dimethyl suberimidate to form a complex which lacks the ability to catalyze the known host functions catalyzed by the individual elongation factors. Using a previously developed reconstitution system we have examined the effects of crosslinking the EF-Tu-Ts complex on reconstituted replicase activity. Renaturation is significantly more efficient when exogenously added native EF-Tu-Ts is crosslinked than when it is not. Crosslinked EF-Tu-Ts can be purified from a crude crosslinked postribosomal supernatant by its ability to replace EF-Tu and EF-Ts in the renaturation of denatured Qbeta replicase. A sample of Qbeta replicase with crosslinked EF-Tu-Ts replacing the individual elongation factors was prepared. Although it lacked EF-Tu and EF-Ts activities, it could initiate transcription of both poly(C) and Qbeta RNA normally and had approximately the same specific activity as control enzyme. Denatured Qbeta replicase formed with crosslinked EF-Tu-Ts was found to renature much more rapidly than untreated enzyme and, in contrast to normal replicase, its renaturation was not inhibited by GDP. The results demonstrate that EF-Tu and EF-Ts function as complex in Qbeta replicase and do not perform their known protein biosynthetic function in the RNA synthetic reaction.
...
PMID:Reconstitution of Qbeta RNA replicase from a covalently bonded elongation factor Tu-Ts complex. 106 92

Escherichia coli Phage Qbeta RNA replicase, an RNA-dependent RNA polymerase, is a tetramer composed of one phage-coded polypeptide and three host-supplied polypeptides which are known to function in the biosynthesis of proteins in the uninfected host. Two of these polypeptides, protein synthesis elongation factors EF-Tu and EF-Ts, are required for initiation of transcription by Qbeta replicase with all templates. Using a previously developed reconstitution system we have examined the effects of modification of EF-Tu on reconstituted replicase activity. The poly(G) polymerase activity of the enzyme can be recovered after pretreatment of the EF-Tu-GDP with either L-1-tosylamido-2-phenylethyl chloromethyl ketone or N-ethylmaleimide, both of which inhibit the aminoacyl-tRNA binding activity of EF-Tu. This suggests that the aminoacyl-tRNA binding site of EF-Tu is not required for Qbeta replicase activity. When Qbeta replicase is treated with kirromycin, an antibiotic which modifies EF-Tu activity by an unknown mechamism, the protein synthetic activity of the EF-Tu in the replicase complex is eliminated but the Qbeta RNA replication activity is only slightly affected. Treatment of pure EF-Tu with kirromycin, however, prevents it from functioning in the renaturation of Qbeta replicase. This antibiotic is not effective against the EF-Tu-Ts complex in the reconstitution assay. Kirromycin at the relatively high concentration used here is found to prevent the formation of the EF-Tu-Ts complex. GDP, which binds to EF-Tu and inhibits formation of the complex with EF-Ts, also inhibits renaturation of Qbeta replicase. It is suggested that the EF-Tu-Ts complex, rather than the individual polypeptides, functions in the renaturation of Qbeta replicase and that the kirromycin and GDP act by preventing formation of this complex.
...
PMID:Function and structure in ribonucleic acid phage Qbeta ribonucleic acid replicase. Effect of inhibitors of EF-Tu on ribonucleic acid synthesis and renaturation of active enzyme. 126 42

In a previous paper we described a number of Escherichia coli mutants resistant to the antibiotic kirromycin. These mutants are altered in both tufA and tufB, the genes coding for elongation factor Tu (EF-Tu). We have now isolated EF-Tu in a homogeneous form from the mutant strains and have studied its function in polypeptide synthesis. These EF-Tu preparations were examined in renaturation studies of Qbeta RNA replicase, described in another paper. In order to characterize the factor we have inactivated the tufB gene by insertion of bacteriophage Mu or by an amber mutation. This enabled us to isolate EF-Tu as a single gene product derived from tufA (designated EF-TuA in contrast to the tufB product, which is called EF-TuB). Kirromycin-resistant EF-TuA did not respond to addition of the antibiotic in three assays: [(3)H]GDP exchange with EF-Tu-GDP at 0 degrees C, in vitro translation of poly(U), and kirromycin-induced GTPase activity of EF-Tu. In contrast, wild-type EF-TuA responded normally to the antibiotic in these assays. One of our mutants (LBE 2012) harbors the kirromycin-resistant EF-TuA and an EF-TuB that is able to bind kirromycin. This binding does not cause inhibition of protein synthesis, indicating that EF-TuB from LBE 2012 is unable to reach the ribosome under these conditions. The two types of EF-Tu from this mutant are equal in size but differ by 0.1 pH unit in isoelectric point. In the soluble fractions of LBE 2012 cells they are present in approximately equal amounts. Our results also show that the tufB gene is not necessary for bacterial growth.
...
PMID:Elongation factor Tu isolated from Escherichia coli mutants altered in TufA and tufB. 700 48

We have isolated an ADP-ribosylation factor (ARF) gene from the human malarial parasite, Plasmodium falciparum. The gene (P. falciparum arf1) has four introns and the exons encode a protein of 181 amino acids with high similarity to the mammalian class I ARF proteins 1-3 (> or = 74% amino acid identity). Southern hybridization suggests there is at least one additional arf in the P. falciparum genome. Northern analysis identified a single P. falciparum arf1 mRNA of 1.8 kb in the asexual blood stage form of the parasite. The P. falciparum arf1 mRNA levels are developmentally regulated, reaching a maximum during nuclear division towards the end of the intraerythrocytic cycle. P. falciparum arf1 cDNA was isolated by reverse-transcriptase polymerase chain reaction and used to express a recombinant protein in Escherichia coli. Recombinant P. falciparum ARF1 protein was purified with stoichiometric amounts of bound GDP, although intrinsic guanose triphosphatase activity of the protein could not be detected. The protein stimulated cholera-toxin-catalyzed ADP-ribosyltransferase activity in a reaction that was dependent upon the addition of both dimyristoylglycerophosphocholine and cholate. The protein bound GTP with first-order kinetics with an apparent rate constant, k', of 0.0145 (+/- 0.0019) min-1. These results suggest that P. falciparum ARF1 is a member of the class 1 ARF family and provide additional evidence for the existence of a classical secretory pathway in P. falciparum.
...
PMID:Isolation, expression and characterization of the gene for an ADP-ribosylation factor from the human malaria parasite, Plasmodium falciparum. 895 60

The brome mosaic virus (BMV) RNA-dependent RNA polymerase (RdRp) directs template-specific synthesis of (-)-strand genomic and (+)-strand subgenomic RNAs in vitro. Although the requirements for (-)-strand RNA synthesis have been characterized previously, the mechanism of subgenomic RNA synthesis has not. Mutational analysis of the subgenomic promoter revealed that the +1 cytidylate and the +2 adenylate are important for RNA synthesis. Unlike (-)-strand RNA synthesis, which required only a high GTP concentration, subgenomic RNA synthesis required high concentrations of both GTP and UTP. Phylogenetic analysis of the sequences surrounding the initiation sites for subgenomic and genomic (+)-strand RNA synthesis in representative members of the alphavirus-like superfamily revealed that the +1 and +2 positions are highly conserved as a pyrimidine-adenylate. GDP and dinucleotide primers were able to more efficiently stimulate (-)-strand synthesis than subgenomic synthesis under conditions of limiting GTP. Oligonucleotide products of 6-, 7-, and 9-nt were synthesized and released by RdRp in 3-20-fold molar excess to full-length subgenomic RNA. Termination of RNA synthesis by RdRp was not induced by template sequence alone. Our characterization of the stepwise mechanism of subgenomic and (-)-strand RNA synthesis by RdRp permits comparisons to the mechanism of DNA-dependent RNA synthesis.
...
PMID:Mechanistic analysis of RNA synthesis by RNA-dependent RNA polymerase from two promoters reveals similarities to DNA-dependent RNA polymerase. 963 Feb 51

NS5B of the hepatitis C virus is an RNA template-dependent RNA polymerase and therefore the key player of the viral replicase complex. Using a highly purified enzyme expressed with recombinant baculoviruses in insect cells, we demonstrate a stimulation of RNA synthesis up to 2 orders of magnitude by high concentrations of GTP but not with ATP, CTP, UTP, GDP, or GMP. Enhancement of RNA synthesis was found with various heteropolymeric RNA templates, with poly(C)-oligo(G)12 but not with poly(A)-oligo(U)12. Several amino acid substitutions in polymerase motifs B, C, and D previously shown to be crucial for RdRp activity were tested for GTP stimulation of RNA synthesis. Most of these mutations, in particular those affecting the GDD motif (motif C) strongly reduced or completely abolished activation by GTP, suggesting that the same NTP-binding site is used for stimulation and RNA synthesis. Since GTP did not affect the overall RNA binding properties or the elongation rate, high concentrations of GTP appear to accelerate a rate-limiting step at the level of initiation of RNA synthesis. Finally, enhancement of RNA synthesis by high GTP concentrations was also found with NS5B of the pestivirus classical swine fever virus, but not with the 3D polymerase of poliovirus. Thus, stimulation of RdRp activity by GTP is evolutionarily conserved between the closely related hepaciviruses and pestiviruses but not between these and the more distantly related picornaviruses.
...
PMID:Selective stimulation of hepatitis C virus and pestivirus NS5B RNA polymerase activity by GTP. 1019 56

LALP70 is a novel lysosomal membrane protein belonging to the apyrase protein family. The apyrase protein family comprises enzymes capable of cleaving nucleotide tri- and diphosphates in a calcium- or magnesium-dependent manner, not being altered by P-type, F-type, or V-type NTPase inhibitors. In this study we have cloned and sequenced the human LALP70 gene to determine the genomic structure. The gene is organized in 11 introns and 12 exons covering a genomic region of approximately 16 kilobase pairs. By fluorescence in situ hybridization analysis, the hLALP70 gene was mapped to the human chromosome 8p21.1-p21.3. We further show that there is at least one alternatively spliced variant, hLALP70v, which can be generated via an alternative splice side at the 3'-end of exon 7, leading to a protein variant differing in 8 amino acids (VSFASSQQ). This is the first splice variant that has been described in the apyrase protein family. Reverse transcriptase polymerase chain reaction analysis showed an ubiquitous expression of both variants, with different relative mRNA expression levels in different tissues. Comparison of the enzymatic properties of the splice variants revealed a broader substrate specificity for hLALP70v with CTP, UDP, CDP, GTP, and GDP as preferred substrates, while hLALP70 utilized UTP and TTP preferentially. Furthermore, enzyme activity of hLALP70v was equally dependent on Ca(2+) and Mg(2+), being saturated already at 1 mm concentration. In contrast, hLALP70 enzymatic activity were unsaturated up to 10 mm Ca(2+), while Mg(2+) showed a saturation at already 1 mm concentration with 2-3-fold lower enzymatic activity as observed with Ca(2+). Our data suggest that the presence or absence of the 8-amino acid motif VSFASSQQ provoke differences in substrate specificity and divalent cation dependence of hLALP70/hLALP70v.
...
PMID:First apyrase splice variants have different enzymatic properties. 1085 52

Phospholipase C (PLC) activity was investigated by stimulation of membrane preparations obtained from insulin (beta-TC3)-, somatostatin (Rin 1027-B2)-, and glucagon (INR1-G9)-producing pancreatic cell lines using the non-hydrolyzable GTP analogue GTPgammaS alone, the C-terminal octapeptide cholecystokinin (CCK-8), or gastrin. All compounds caused a significant 2- to 4.4-fold stimulation of PLC activity in the different cell lines, which was diminished by the non-hydrolyzable GDP analogue GDPbetaS. CCK receptor subtypes were characterized by radioligand binding experiments. High-affinity binding sites for tritiated CCK(A) receptor antagonist L-364,718 (K(d) = 0.24 nM) and tritiated CCK(B) receptor antagonist L-365,260 (K(d) = 0.13 nM) were only present in Rin 1027-B2 cells. High-affinity binding sites for both ligands were not found in beta-TC3 or INR1-G9 cells. Competition binding experiments with non-labeled CCK receptor antagonists CR 1505 (CCK(A) receptor-selective) and CR 2945 (CCK(B) receptor-selective), as well as microphysiometry experiments, resulted in the same receptor distribution. Reverse transcriptase-polymerase chain reaction confirmed the CCK receptor distribution pattern for Rin 1027-B2 cells, but in addition showed the existence of CCK(B) receptors in beta-TC3 cells. Immunoblocking experiments with C-terminal antibodies against different G-protein alpha-subunits demonstrated inhibition of CCK-stimulated PLC activity in beta-TC3 cells by G(q/11)alpha antiserum (70%), in Rin 1027-B2 cells by G(q/11)alpha antiserum (70%) and G(i)-3alpha antiserum (23%), and in INR1-G9 cells by G(q/11)alpha antiserum (60%) and G(o)alpha antiserum (45%). We conclude that CCK receptor subtypes with different G-protein-coupling specificities to PLC are present in the different hormone-secreting cells of the endocrine pancreas.
...
PMID:Activation of phospholipase C by cholecystokinin receptor subtypes with different G-protein-coupling specificities in hormone-secreting pancreatic cell lines. 1093 May 42

To understand primary cell wall assembly in Arabidopsis, we have focused on identifying and characterizing enzymes involved in xyloglucan biosynthesis. Nine genes (AtFUT2-10) were identified that share between 47% and 62% amino acid similarity with the xyloglucan-specific fucosyltransferase AtFUT1. Reverse transcriptase-PCR analysis indicates that all these genes are expressed. Bioinformatic analysis predicts that these family members are fucosyltransferases, and we first hypothesized that some may also be involved in xyloglucan biosynthesis. AtFUT3, AtFUT4, and AtFUT5 were expressed in tobacco (Nicotiana tabacum L. cv BY2) suspension culture cells, and the resulting proteins did not transfer fucose (Fuc) from GDP-Fuc to tamarind xyloglucan. AtFUT3, AtFUT4, and AtFUT5 were overexpressed in Arabidopsis plants. Leaves of plants overexpressing AtFUT4 or AtFUT5 contained more Fuc than wild-type plants. Stems of plants overexpressing AtFUT4 or AtFUT5 contained more xylose, less arabinose, and less galactose than wild-type plants. We suggest that the AtFUT family is likely to include fucosyltransferases important for the synthesis of wall carbohydrates. A targeted analysis of isolated cell wall matrix components from plants altered in expression of these proteins will help determine their specificity and biological function.
...
PMID:Characterization of a family of Arabidopsis genes related to xyloglucan fucosyltransferase1. 1174 4


1 2 3 Next >>

---