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

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

We have studied the effect of protein phosphokinase (EC 2.7.1.37; ATP:protein phosphotransferase) and phosphoprotein phosphatase (EC 3.1.3.16; phosphoprotein phosphohydrolase) on reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) activity of Rous sarcoma virus. Protein kinase from Rous sarcoma virus-transformed chick embryo fibroblasts was purified by DEAE-cellulose chromatography, Sephadex gel filtration, and isoelectric focusing. Purified reverse transcriptase from Rouse sarcoma virus was preincubated with protein kinase and ATP under conditions allowing incorporation of phosphate into substrate protein. After the preincubation, reverse transcriptase activity was assayed in the presence of poly(rA).oligo(dT) as template. A 2- to 5-fold increase of reverse transcriptase activity was found after the preincubation of reverse transcriptase with protein kinase and ATP. Incubation of reverse transcriptase with heat-treated, inactive protein kinase and ATP had no effect on transcriptase activity. When the transcriptase preparation was incubated with protein kinase and [gamma-32P]ATP and subsequently purified by chromatography on phosphocellulose and Sephadex gel filtration, significant amounts of 32P-labeled proteins were found in the fractions exhibiting reverse transcriptase activity, suggesting 32P incorporation into transcriptase or transcriptase-associated proteins. A 20-60% decrease of reverse transcriptase activity was observed after incubation of reverse transcriptase with phosphatase. The results suggest that phosphorylative modification of reverse transcriptase may be critical in the regulation of reverse transcriptase-catalyzed DNA synthesis.
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PMID:Protein kinase and its regulatory effect on reverse transcriptase activity of Rous sarcoma virus. 5 72

The ATP requirement during RNA transcription by the RNA-dependent RNA polymerase associated with purified vesicular stomatitis virus was studied during chain initiation and chain elongation steps. Initiation of transcription in vitro has an apparent Km for ATP of approximately 500 micron, whereas the apparent Km for ATP during chain elongation is almost identical with those for the other three ribonucleoside triphosphates, i.e. 20 to 30 micron. Further analysis of the transcription process demonstrated that the beta-gamma imido analogue of ATP cannot be used for the initiation step. However, it can be incorporated into RNA during chain elongation. These results indicate that a hydrolyzable form of the beta-gamma phosphodiester bond in ATP is required for the initiation process and also support the hypothesis for a single promoter site for the initiation of transcription on the genome of vesicular stomatitis virus.
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PMID:Initiation of RNA synthesis in vitro by vesicular stomatitis virus. Role of ATP. 21 77

Adenosine (beta,gamma-imido)triphosphate (AMP-PNP) and guanosine (beta,gamma-imido)triphosphate (GMP-PNP) are analogs of ATP and GTP with non-hydrolyzable gamma-phosphates. Although both AMP-PNP and GMP-PNP were used in place of ATP and GTP by Escherichia coli RNA polymerase to transcribe vaccinia virus DNA, only GMP-PNP was used by the transcriptase present within vaccinia virus cores. AMP-PNP specifically prevented initiation of transcription, since RNA initiated in the presence of ATP, GTP, and CTP was subsequently elongated by incubating the washed cores in the presence of AMP-PNP, GTP, CTP, and UTP. The RNA formed in this manner, however, was (i) several times longer than normal transcripts, indicating a defect in chain termination and/or cleavage of nascent RNA, (ii) was not polyadenylylated (although free polyadenylic acid formed), and (iii) was not extruded from the virus cores. Nearest neighbor analysis demonstrated that AMP-PNP was incorporated adjacent to all four nucleotides, and hybridization to restriction endonuclease fragments of vaccinia virus DNA indicated that the high-molecular-weight RNA was transcribed from representative fractions of the entire genome. The possibility of a block in processing rather than or in addition to a block in chain termination was suggested by the cleavage of the high-molecular-weight RNA within the core after replacement of AMP-PNP with ATP. Cleavage of purified high-molecular-weight RNA by a soluble endoribonuclease extracted from vaccinia virus cores, however, was not dependent upon ATP, nor was it inhibited by AMP-PNP. The latter results suggest that AMP-PNP blocks a step preceding cleavage.
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PMID:Multiple roles for ATP in the synthesis and processing of mRNA by vaccinia virus: specific inhibitory effects of adenosine (beta,gamma-imido) triphosphate. 69 Nov 15

The reactivity of the sulfhydryl groups of histidyl-t RNA synthetase from Salmonella typhimurium and the effect of substrates on the reactivity has been studied using p-hydroxymercuribenzoate and 5, 5'-dithiobis (2-nitrobenzoic acid) as reagents. It has been found that 5, 5'-dithiobis (2-nitrobenzoic acid) titrates only two sulfhydryl groups per molcule of enzyme and the reaction is essenaitlly monophasic, while p-hydroxymercuribenzoate titrates four sulhydryl groups. As observed kinetically the reaction with p-hydroxymercuribenzoate is strongly biphasic, each phase corresponding to about two sulfhydryl groups per enzyme molecule. With both reagents no detectable difference in sulfhydryl group reactivity was observed when ATP, histidine and tRNA specific for histidine were added individually or in combination to the enzyme. The enzyme activity slowly changes after two or four sulhydryl groups are blocked by 5, 5'-dithiobis (2-nitrobenzoic acid) or p-hydroxymercuribenzoate respectively. A new, stable level of activity is reached that is characterized by a different Km value for the aminoacylation reaction. The results indicate that the sulfhydryl groups reacting with the two reagents used here are neither directly involved in the binding of the substrates nor in the catalytic process. The ultimate change in enzyme activity after reaction of the sulfhydryl groups suggests a transition to an alternative enzyme structure.
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PMID:Histidyl-transfer-ribonucleic-acid synthetase from Salmonella typhimurium. Studies of the sulfhydryl groups. 77 25

Hepatitis is transmitted by a number of infectious agents. The epidemiological characterization of waterborne or enterically transmitted non-A, non-B hepatitis (ET-NANBH) is unique when compared with other known hepatitides. We have reported on the molecular cloning of a cDNA clone derived from the etiologic agent associated with ET-NANBH, the hepatitis E virus (HEV). The complete sequence of these first molecular clones, isolated from an HEV-infected human after passage in Macaca fascicularis (cynomolgus macaques), illustrates a distant relationship to other known positive-strand RNA viruses of plants and animals. The translated major open reading frame (ORF-1) from these clones indicates that this portion of the genome encodes a polyprotein with consensus sequences found in RNA-dependent RNA polymerase and ATP/GTP binding domains. The latter activity has been associated with putative helicases of positive-strand RNA viruses. These viral-encoded enzymatic activities identify this region and ORF-1 as containing at least two different nonstructural genes involved in HEV replication. Molecular clones obtained from two other geographically distinct HEV isolates demonstrated sequence heterogeneity in this nonstructural gene region. Further study will be required to elucidate the pathogenic significance (if any) of this observed divergence in the nonstructural region.
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PMID:Hepatitis E virus (HEV): strain variation in the nonstructural gene region encoding consensus motifs for an RNA-dependent RNA polymerase and an ATP/GTP binding site. 158 64

Rotavirus single-shelled particles have several enzymatic activities that are involved with the synthesis of capped mRNAs both in vivo and in vitro. Because single-shelled particles must be structurally intact to carry out transcription, it has proven to be difficult to identify the protein within such particles that possesses associated RNA polymerase activity. One approach for characterizing the function of the individual proteins within single-shelled particles is to use nucleotide analogs to specifically label those proteins, such as the viral RNA polymerase, that have affinity for nucleotides. In this study, 8-azido-ATP (azido-ATP), a photoreactable nucleotide analog, was used to identify the viral RNA polymerase on the basis of the ability of the analog to inhibit transcription activity associated with rotavirus particles on exposure to UV light. When single-shelled particles were treated with UV light in the presence of [alpha-32P]azido-ATP, the structural protein VP1 became radiolabeled because of cross-linking of the nucleotide analog, and there was a corresponding decrease in the ability of the particles to synthesize mRNA. In parallel experiments in which single-shelled particles were not exposed to UV light, VP1 was not radiolabeled and the particles successfully used azido-ATP as a substrate for the synthesis of viral mRNAs. Taken together, these results are consistent only with the conclusion that VP1 is the rotavirus RNA-dependent RNA polymerase.
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PMID:Photoaffinity labeling of rotavirus VP1 with 8-azido-ATP: identification of the viral RNA polymerase. 164 6

The refined crystal structure of Escherichia coli glutaminyl transfer RNA synthetase complexed with transfer RNA(Gln) and ATP reveals that the structure of the anticodon loop of the enzyme-bound tRNA(Gln) differs extensively from that of the known crystal structures of uncomplexed tRNA molecules. The anticodon stem is extended by two non-Watson-Crick base pairs, leaving the three anti-codon bases unpaired and splayed out to bind snugly into three separate complementary pockets in the protein. These interactions suggest that the entire anticodon loop provides essential sites for glutaminyl tRNA synthetase discrimination among tRNA molecules.
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PMID:Structural basis of anticodon loop recognition by glutaminyl-tRNA synthetase. 185 17

This study has investigated the characteristics of a leucine aminoacyl transfer RNA synthetase enzyme from Tritrichomonas augusta. Differential centrifugation and DEAE-cellulose column chromatography were used for partial enzyme purification. The column purification increased the synthetase activity 125-fold over the unfractionated cell extract. The conditions for maximum [3H] leucine charging were 37 degrees C for 20 min, with protein at 180 micrograms ml-1 using yeast leucine tRNA as an acceptor. The optimal reaction conditions were 14 mM-Mg acetate, 3 mM-ATP, 3 mM-spermidine and 5.5 mM-putrescine. Acceptor activity with T. augusta transfer RNA was 8-fold higher than with yeast transfer RNA and 25-fold higher than with Escherichia coli transfer RNA. The partially purified enzyme fraction had comparable changing activities for both leucine and valine.
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PMID:Characteristics of a leucine aminoacyl transfer RNA synthetase from Tritrichomonas augusta. 186 66

A portion of the RNA genome of beet yellows closterovirus (BYV) has been sequenced encompassing a complete long open reading frame (ORF) potentially encoding a 65 kDa protein. The sequence of this putative protein was strikingly similar to those of HSP70-related heat shock proteins. The counterparts of all the eight segments strongly conserved in HSP70s could be confidently identified in the BYV 65 kDa protein. It is suggested that some of these segments might be the ATP-binding site(s) and that, similarly to the heat shock proteins, the 65 kDa is probably ATP-binding. Generally, however, the divergence between the 65 kDa sequence and the sequences of the HSP70s was much more pronounced than that between any two members of the latter family, allowing a clearer delineation of clusters of conserved residues that might be crucial for protein function. It is suggested that these observations will be helpful in functional dissection of the proteins of the HSP70 family. Analysis of the sequence of a portion of the ORF found upstream from the 65 kDa ORF showed that the C-terminal domain of the encoded protein could be an RNA-dependent RNA polymerase closely related to those of tricornaviruses, a family of RNA plant viruses with three component genomes.
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PMID:Putative 65 kDa protein of beet yellows closterovirus is a homologue of HSP70 heat shock proteins. 200 13

Cytoplasmic extracts made from HeLa cells that have been harvested late after infection with vaccinia virus are capable of specifically transcribing templates containing vaccinia virus late-gene promoters. We applied such an extract to a phosphocellulose column and eluted the proteins with a series of buffers containing successively higher concentrations of NaCl. None of three column fractions alone was capable of specific transcription of a late-gene template. However, specific transcriptase activity could be reconstituted by mixing column fractions, with maximal activity seen when all three fractions were present. The activities present in all fractions were heat labile, resistant to micrococcal nuclease, and present only in extracts from vaccinia virus-infected cells. A quantitative complementation assay was used to further purify one factor, named VLTF-1, over subsequent columns of DEAE-cellulose and hydroxylapatite. VLTF-1 was separated from endogenous RNA polymerase, was a late-promoter-specific transcription factor, and had a sedimentation rate consistent with an apparent Mr of 45,000. The RNA polymerase-containing fraction was not only necessary for transcription with a late-promoter template but alone was capable of specifically transcribing a vaccinia virus early-gene promoter. A further difference between early and late gene transcription in this system was in the ability of the ATP analog beta-8-imidoadenosine-5'-triphosphate (AMP-PNP) to substitute for ATP in supporting specific transcription of only the late-promoter template. The system reconstituted from the various fractions retained the ability to produce the novel poly(A) sequence found on the 5' end of vaccinia virus late messages.
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PMID:Identification of factors specific for transcription of the late class of vaccinia virus genes. 247 68


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