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)

Much work has been done on the isolation, purification, and characterization of the RNA-directed RNA polymerase (EC 2.7.7.48) of cucumber mosaic virus (CMV)-infected cucumbers. Uninfected plants were reported to have no such enzyme, but we recently detected low levels of the activity in cucumber. Since tobacco and cowpea contain such an enzyme that is variably increased in amount by various virus (as well as viroid) infections, we assumed that this would also be the case upon CMV infection of cucumber. However, further purification and characterization of the RNA-directed RNA polymerases from healthy and from infected cucumber suggests that these are different enzymes. The presumed CMV replicase was obtained pure and consists of a major polypeptide of Mr 100,000 and minor components of Mr 110,000 and about 10,000. The Km is 5 microM ([3H]GTP) when tobacco mosaic virus RNA is used as template.
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PMID:RNA-directed RNA polymerases from healthy and from virus-infected cucumber. 345 3

An RNA-dependent RNA polymerase (replicase) extract from brome mosaic virus-infected barley leaves has been shown to initiate synthesis of (-) sense RNA from (+) sense virion RNA. Initiation occurred de novo, as demonstrated by the incorporation of [gamma-32P]GTP into the product. Sequencing using cordycepin triphosphate to terminate (-) strands during their synthesis by the replicase generated sequence ladders that confirmed that copying was accurate, and that initiation occurred very close to the 3' end. The precise site of initiation was further defined by testing the replicase template activity after stepwise removal of 3'-terminal nucleotides. Whereas removal of the terminal A did not decrease template activity, removal of the next nucleotide (C-2) did. Thus, initiation almost certainly occurs opposite the penultimate 3'-nucleotide (C-2) in vitro. The structure of the double-stranded replicative form of RNA isolated from brome mosaic virus-infected leaves was consistent with such a mechanism occurring in vivo, in that it lacked the 3'-terminal A found on virion RNAs. The specific site of (-) strand initiation and normal template activity were retained for RNAs with as many as 15 to 30 A residues added to the 3' end. However, only limited oligonucleotide 3' extensions can be present on active templates. In order to assess the 5' extent of sequences required for an active template, a 134-nucleotide-long fragment of brome mosaic virus RNA, corresponding to the tRNA-like structure, was generated. This RNA had high template activity, but a shorter 3' (85-nucleotide) fragment was inactive. RNAs with various heterologous sequences 5' to position 134 also showed high template activity. Thus, the 3'-terminal tRNA-like structure common to all four brome mosaic virus virion RNAs contains all of the signals required for initiation of replication, and sequences 5' to it do not play a role in template selection.
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PMID:Minus-strand initiation by brome mosaic virus replicase within the 3' tRNA-like structure of native and modified RNA templates. 375 4

Previous data demonstrated that reovirus mRNA, synthesized in vitro with the particulate RNA transcriptase of reovirus cores, efficiently directs the synthesis of polypeptides in vitro. The present studies indicate that all of the three size classes of reovirus mRNA produced in vitro can form protein initiation complexes with rat liver [(36)S]Met-tRNA(F) and incubated 40S and 60S ribosomal subunits, which had been washed in 0.5 M KCl of mouse fibroblast L-929 cells. Mild prior treatment of the mRNA with HCHO was required to expose the initiator region. The initiation complex reacted quantitatively with puromycin to form a puromycin peptide, whose electrophoretic properties were identical to methionyl-puromycin formed in response to poly(A,G,U) or the initiator codon AUG. The complex was relatively stable and specific for [(35)S]Met-tRNA(F); rat liver [(35)S]Met-tRNA(M) was unreactive unless the supernatant factors EF T(1) and EF T(2) were also present. However, the addition of fusidic acid, at a concentration that did not affect complex formation with [(35)S]Met-tRNA(F), completely inhibited Met-tRNA(M) utilization. Exogenous ribosomal factors and GTP were not required unless the separated 40S and 60S subunits were further treated with 1 M KCl. The data suggest that reovirus mRNA contains AUG initiator codons that form a complex with Met-tRNA(F) at a puromycin-reactive site on ribosomes.
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PMID:Formation of a mammalian initiation complex with reovirus messenger RNA, methionyl-tRNA F , and ribosomal subunits. 450 35

The enzymes responsible for replication of the RNA of the single-stranded RNA bacteriophages contain, in addition to one phage-coded polypeptide, three host-coded polypeptides taken from the protein biosynthetic machinery: ribosomal protein S1 and the elongation factors Tu and Ts. While S1 performs a function in RNA replication derived from its protein synthetic function, mRNA binding, the reactions catalysed by the elongation factors in protein synthesis are apparently dispensible for RNA replication. In the replicase, these polypeptides, acting as the EF-Tu . Ts complex, play a fundamental structural role. Replacement of the endogenous EF-Tu with mutant EF-Tu, itself stable, causes the RNA replicase to become unstable. The possibility that EF-Tu . Ts is solely a structural protein in the RNA replicase is suggested by experiments showing that a variety of modifications of the elongation factors can be tolerated without loss of RNA synthetic capacity. In fact, EF-Tu . Ts from distantly related bacterial species can substitute for E. coli EF-Tu . Ts in RNA replicase. Evidence is presented that the high in vitro template specificity of Q beta replicase may be accomplished through modulation of the level of GTP required for initiation of transcription. Different natural and synthetic RNAs require quite different GTP concentrations. Mn2+ ions, which extend the range of templates transcribed by Q beta replicase, lower the requirement for GTP. High ionic strength, which alters the conformation of Q beta replicase such that template specificity is increased, raises the GTP requirement. An additional host coded protein required for in vitro Q beta RNA replication, host factor (HF), interacts specifically with Q beta RNA. This polypeptide acts by allowing Q beta replicase to initiate RNA synthesis with Q beta RNA at reduced GTP concentration.
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PMID:Interaction of host-coded and virus-coded polypeptides in RNA phage replication. 610 96

Pyridoxal 5'-phosphate (PLP), a reversible inhibitor of in vitro transcription by fowl plaque virus, has been used to identify the transcriptase. Kinetic analyses showed that PLP competitively inhibits the addition of each nucleoside triphosphate in ApG-primed reactions, suggesting that both initiation and elongation are affected. The irreversible inhibition by PLP following reduction with borohydride was prevented by preincubation with the first substrate: GTP in unprimed reactions or CTP in the presence of ApG. On reaction of FPV proteins with PLP and [3H]borohydride the core protein PB1 was preferentially labeled and the labeling was selectively blocked by GTP or ApG + CTP. These data suggest that PB1 has the nucleotide-binding site of the transcriptase, is responsible for both initiation and elongation, and is apparently associated with the 3' ends of template RNAs in virions.
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PMID:Identification of the influenza virus transcriptase by affinity-labeling with pyridoxal 5'-phosphate. 619 1

We propose a mechanism for the priming of influenza viral RNA transcription by capped RNAs in which specific 5'-terminal fragments are cleaved from the capped RNAs by a virion-associated endonuclease. These fragments would serve as the actual primers for the initiation of transcription by the initial incorporation by the initial incorporation of a G residue at their 3' end. We show that virions and purified viral cores contain a unique endonuclease that cleaves RNAs containing a 5' methylated cap structure (m7GpppXm) preferentially at purine residues 10 to 14 nucleotides from the cap, generating fragments with 3'-terminal hydroxyl groups. RNAs containing the 5'-terminal structure GpppG could not be cleaved to produce these specific fragments. Consistent with our proposed mechanism, those capped fragments that function as primers could be linked to a G residue in transcriptase reactions containing alpha-32P-GTP as the only ribonucleoside triphosphate. The pattern of G and C incorporation onto these primer fragments suggests that this incorporation is directed by the second and third bases at the 3' end of the virion RNA template, which has the sequence 3' UCG. Primer fragments with a 3'-terminal A residue were used more efficiently than those with a 3'-terminal G residue, indicating a preference for generating an AGC sequence in the viral mRNA complementary to the 3' end of the virion RNA. Cleavage of the RNA primer and initiation of transcription are not necessarily coupled, because a 5' fragment isolated from one reaction could be used as a primer when added to a second reaction. Uncapped ribopolymer inhibitors of viral RNA transcription inhibited the cleavage of capped RNAs.
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PMID:A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. 626 60

To identify the initial steps of vesicular stomatitis virus transcription, we reconstituted purified nucleocapsid template with solubilized transcriptase and characterized the in vitro products of de novo transcription. In the absence of UTP and GTP, only leader gene products were synthesized; mRNA oligonucleotides were detected only after transcription of full-length leader was permitted. These data suggest that vesicular stomatitis virus polymerase does not enter the genome independently at each gene, but each polymerase begins transcription at the 3' end of the genome, and reaches internal genes only by sequentially transcribing the 3' preceding sequences. These results are consistent with the conclusion that the observed sequential transcription of vesicular stomatitis virus mRNAs is due to obligatory entrance of all polymerases at the leader gene, and suggest that the transcriptase and replicase may recognize the same promoter.
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PMID:Reconstitution studies detect a single polymerase entry site on the vesicular stomatitis virus genome. 629 77

Three double-stranded RNA segments of bacteriophage phi 6 (L, M, and S) were transcribed in vitro by a virion-associated RNA polymerase. Regulation of L transcription was distinct from regulation of M and S transcription. Transcription of the L segment, which codes for early proteins, required manganous ion and high concentrations of all four ribonucleoside triphosphates and was inhibited by polyamines such as spermine. Transcription of the M and S segments, which code for late proteins, required manganous or magnesium ion and relatively low concentrations of all ribonucleoside triphosphates except GTP and was enhanced by polyamines. Optimal conditions for L transcription were more stringent than those for M and S transcription. These two apparently different patterns produced in in vitro transcription presumably reflect the two distinct in vivo transcription patterns; i.e., (i) similar amounts of three single-stranded RNA species were transcribed from the three corresponding segments of double-stranded RNA (early pattern) and (ii) a much larger amount of single-stranded RNA species was transcribed from M and S segments than from the L segment (late pattern). The early transcription pattern may be changed into the late pattern by a change of environment, such as substrate concentration. This suggests that the different enzymatic properties under the different environmental conditions of the virion-associated transcriptase are responsible for the transcriptional regulation throughout the infection cycle of bacteriophage phi 6.
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PMID:Transcriptional regulation of three double-stranded RNA segments of bacteriophage phi 6 in vitro. 682 50

One of the four subunits of bacteriophage Q beta RNA replicase is elongation factor Tu (EF-Tu), the host aminoacyl-tRNA (AA-tRNA) binding protein. To determine whether the RNA polymerase activity requires the tRNA binding site of EF-Tu, we reconstituted replicase with EF-Tu . GTP covalently bound to AA-tRNA. This cross-linked ternary complex (XLTC) was formed by the reaction of N epsilon-bromoacetyl-Lys-tRNA with EF-Tu-GTP. In an EF-Tu-dependent system for the reconstitution of replicase, XLTC restored polymerase activity at least as well as an equivalent amount of EF-Tu. Replicase reconstituted with XLTC was resolved from replicase containing EF-Tu by chromatography on phosphocellulose, a result which confirmed that the tRNA moiety was incorporated into the enzyme. Chromatographic analysis of reconstitution mixtures revealed that XLTC was incorporated into replicase as extensively as EF-Tu. From these results, it appears that the AA-tRNA binding site on EF-Tu is not required for the assembly or activity of Q beta RNA replicase. Furthermore, because the tRNA macromolecule is cross-linked to His-66 of the EF-Tu, the region surrounding His-66 must normally be exposed on the surface of the replicase.
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PMID:Transfer RNA cross-linked to the elongation factor Tu subunit of Q beta replicase does not inhibit Q beta RNA replication. 701 63

The basophilic leucaemia cell line RBL-2H3 exhibits a robust inwardly rectifying potassium current, IKIR, which is likely to be modulated by G proteins. We examined the physiological and molecular properties of this KIR conductance to define the nature of the underlying channel species. The macroscopic conductance revealed characteristics typical of classical K+ inward rectifiers of the IRK type. Channel gating was rapid, first order (tau approximately 1 ms at -100 mV) and steeply voltage dependent. Both activation potential and slope conductance were dependent on extracellular K+ concentration ([K+]o) and inward rectification persisted in the absence of internal Mg2+. The current was susceptible to a concentration- and voltage-dependent block by extracellular Na+, Cs+ and Ba2+. Initial IKIR whole-cell amplitudes as well as current rundown were dependent on the presence of 1 mM internal ATP. Perfusion of intracellular guanosine 5'-Q-(3-thiotriphosphate) (GTP[gamma S]) suppressed IKIR with an average half-time of decline of approximately 400 s. It was demonstrated that the dominant IRK-type 25 pS conductance channel was indeed suppressed by 100 microM preloaded GTP[gamma S]. Reverse transcriptase-polymerase chain reactions (RT-PCR) with RBL cell poly(A)+ RNA identified a full length K+ inward rectifier with 94% base pair homology to the recently cloned mouse IRK1 channel. It is concluded that RBL cells express a classical voltage-dependent IRK-type K+ inward rectifier RBL-IRK1 which is negatively controlled by G proteins.
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PMID:Physiological and molecular characterization of an IRK-type inward rectifier K+ channel in a tumour mast cell line. 760 35


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