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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Temperature-sensitive (ts) mutants of Sindbis virus belonging to complementation group F, ts6, ts110, and ts118, are defective in RNA synthesis at the nonpermissive temperature. cDNA clones of these group F mutants, as well as of ts+ revertants, have been constructed. To assign the ts phenotype to a specific region in the viral genome, restriction fragments from the mutant cDNA clones were used to replace the corresponding regions of the full-length clone Toto1101 of Sindbis virus. These hybrid plasmids were transcribed in vitro by SP6 RNA polymerase to produce infectious transcripts, and the virus recovered was tested for temperature sensitivity. After the ts lesion of each mutant was mapped to a specific region of 400 to 800 nucleotides by this approach, this region of the cDNA clones of both the ts mutant and ts+ revertants was sequenced in order to determine the precise nucleotide change and amino acid substitution responsible for each mutation. Rescued mutants, which have a uniform background except for one or two defined changes, were examined for viral RNA synthesis and complementation to show that the phenotypes observed were the result of the mutations mapped. ts6 and ts110 had a single base substitution in nsP4, resulting in replacement of Gly by Glu at position 153 or position 324, respectively. It is of interest that nsP4 contains the Gly-Asp-Asp motif characteristic of a number of viral replicases, and this, together with the fact that all RNA synthesis in ts6-infected cells and, to a lesser extent, in ts110-infected cells shut off when the cells were shifted from a permissive to a nonpermissive temperature, suggests that nsP4 is the virus polymerase. ts118 was a double mutant. It contained a single base substitution in nsP2, resulting in replacement of Val by Ala at position 425 that resulted in the formation of minute plaques, but not in a reduction in the plaque number at the nonpermissive condition. The second change, a substitution of Gln by Arg in ts118 at residue 93 in nsP4, had little apparent phenotype on its own, but in combination with the change in nsP2 led to a ts phenotype. Thus, in each case the mutation responsible for the temperature sensitivity of the three known complementation group F mutants lay in nsP4. In addition, the result with ts118 suggests that nsP2 and nsP4 may interact with each other in a complex.
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PMID:Mapping of RNA- temperature-sensitive mutants of Sindbis virus: complementation group F mutants have lesions in nsP4. 252 74

An in vitro T7 bacteriophage transcription system has been utilized in which the RNA was initiated to a specific length (defined by the absence of the appropriate nucleoside triphosphate). When the DNA-RNA-RNA polymerase ternary complex was exposed to nonsaturating levels of DNA-binding ligands (i.e., a small fractional occupancy at each site), and the RNA transcript then allowed to elongate in the presence of all four nucleoside triphosphates, there was a synchronous increase of RNA lengths up to sites occupied by ligands. A unique characteristic is that bacteriophage transcription was completely terminated at every ligand site, in contrast to bacterial RNA polymerases where "read-through" past drug sites occurs and results merely in a delay of transcription at each site due primarily to dissociation of drug from the DNA. Similar termination of transcription at each drug site was observed with T3 and SP6 RNA polymerases. The termination at drug sites in the bacteriophage system results in RNA of specific lengths which define the location of ligand sites, and the RNA concentration provides a measure of relative ligand occupancy at that site. Termination of transcription was observed with four drugs with relatively long DNA residence times (half-life greater than or equal to 300 s at 20 degrees C for nogalamycin, actinomycin, mithramycin, and echinomycin) but to a lesser extent with drugs of intermediate residence times [a bis(thiadaunomycin) and an acridine-tripyrrole, with half-lives of 230 and 7 s, respectively, at 20 degrees C]
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PMID:Sequence-dependent termination of bacteriophage T7 transcription in vitro by DNA-binding drugs. 252 55

The processing of the Sindbis virus nonstructural polyprotein translated in vitro has been studied. When Sindbis virus genomic RNA was translated in a reticulocyte lysate, polyprotein P123 was cleaved efficiently to produce nsP1, nsP2, and nsP3. Inhibition of this processing by anti-nsP2 antibodies, but not by antibodies specific for nsP1, nsP3, or nsP4, suggested that the viral proteinase was present in nsP2. To localize the proteolytic activity more precisely, deletions were made in a full-length cDNA clone of Sindbis virus, and RNA was transcribed from these constructs with SP6 RNA polymerase and translated in vitro. Although virtually all of the nsP1, nsP3, and nsP4 sequences could be deleted without affecting processing, deletions in the N-terminal half of nsP2 led to aberrant processing, and deletions in the C-terminal half abolished proteolysis. However, inactive polyproteins containing the nsP2 deletions could be processed by exogenously supplied proteins translated from virion RNA, demonstrating that cleavage was virus specific and not due to a protease present in the reticulocyte lysate and that the deleted polyproteins still served as substrates for the enzyme. From these results and from experiments in which processing was studied at increasingly higher dilution, we have concluded the following: (i) the viral nonstructural proteinase is located in the C-terminal half of nsP2; (ii) in the P123 precursor the cleavage between nsP2 and nsP3 occurs efficiently as a bimolecular reaction (in trans) to remove nsP3, while the bond between nsP1 and nsP2 is cleaved inefficiently, but detectably, in trans, but no autoproteolysis of P123 was detected; (iii) once nsP3 has been removed, the bond between nsP1 and nsP2 in the P12 precursor is cleaved efficiently by autoproteolysis (in cis). This mode of processing leads to a slow rate of cleavage, particularly early in infection, suggesting that the polyproteins might play roles in virus RNA replication distinct from those of the cleaved products. A hypothesis is presented that the proteinase is a thiol protease related to papain.
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PMID:Processing the nonstructural polyproteins of sindbis virus: nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and in trans. 252 79

Subunit-specific DNA probes for the chicken muscle acetylcholine receptor have been used in conjunction with Northern blots and dot-blots based upon the SP6/T7 RNA polymerase reaction to quantitate changes in the steady-state mRNA levels of all four subunits during development. In pectoral muscle, maximal subunit transcript levels are observed at day 12 in ovo for alpha, days 12-16 in ovo for beta, day 14 in ovo for gamma, and days 12-14 in ovo for delta. Interestingly, two delta-subunit transcripts which have somewhat different patterns of temporal expression are detected. At the peak of expression of each subunit mRNA the absolute levels of the alpha-, beta-, and gamma-subunit transcripts are very similar (49.2, 66.0, and 70.7 attomoles of transcript/micrograms of poly(A)+ RNA, respectively) whereas that for the summed delta-subunit transcripts is significantly lower (17.8 attomoles/micrograms of poly(A)+ RNA). Further, this lower level of delta-subunit mRNA is observed from day 10 to day 16 of embryonic development. We conclude that subunit transcript availability is important in the synthesis of acetylcholine receptor protein during development and that the level of the delta-subunit transcript can be rate limiting in the expression of this receptor.
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PMID:Development expression of the genes encoding the four subunits of the chicken muscle acetylcholine receptor. 258 13

Human erythrocytes contain a membrane protein, MACIF, which inhibits the formation of a membrane attack complex (MAC) of complement. We have cloned and sequenced the complementary DNA of MACIF messenger RNA. The amino acid sequence predicted from its nucleotide sequence consists of 128 amino acids. The amino-terminal 25 residues may correspond to a signal peptide. The carboxy-terminal sequence confirmed that MACIF is a glycosylphosphatidylinositol (GPI)-anchored protein. The amino acid sequence of MACIF was partially determined by established techniques for protein chemistry and the resultant sequence was consistent with that predicted from the nucleotide sequence. The results of sequence analyses also suggested that asparagine at the 18th position was N-glycosylated. When mRNA obtained from the MACIF cDNA clone with SP6 RNA polymerase was microinjected into Xenopus oocytes, the oocytes synthesized a product which exhibited MACIF activity and reacted with anti-MACIF antibody. Comparison of the predicted sequence revealed significant homology with mouse Ly-6 antigens.
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PMID:Molecular cloning and characterization of MACIF, an inhibitor of membrane channel formation of complement. 260 9

In studies of the effects of changes in mRNA structure and sequence on the initiation of protein synthesis, we used a generally applicable approach to transcribe reconstructed genes for duck alpha A globin by the bacteriophage SP6 RNA polymerase promoter in a pGEM-2 plasmid vector. The genes were reconstructed such that the first nucleotide to be transcribed, the 5' adenosine, was placed directly adjacent to the SP6 promoter sequence. The 3' ends of the genes were constructed such that cleavage with Ssp 1 endonuclease yielded a template that directed the synthesis of mRNA terminating in a poly A tail containing 56 adenosines and a single 3' uridine. Special conditions using a Mn++ buffer were developed to enable the SP6 RNA polymerase to initiate at the 5' adenosine and synthesize the A-start transcription product. The mRNA could be capped and was subsequently used as an effective template for in vitro translation and synthesis of duck alpha A globin.
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PMID:Construction of mRNA genes for the synthesis and translation of duck alpha globin mRNA. 263 91

A new approach to synthesis of oligoribonucleotides is suggested, based on transcription by E. coli RNA polymerase of synthetic immobilized DNA-templates with AUG as primer. The approach has been experimentally verified by synthesis of two oligonucleotides, viz., a RNA fragment of the fr phage (16 nucleotides long) and a RNA fragment of the tickborne encephalitis virus (18 nucleotides long). Fraction of the synthesized RNA fragments in the whole nucleotide material is about 20%. The templates can be used repeatedly. Sequences of the oligoribonucleotides were confirmed. Advantages of this approach and its usefulness for SP6 DNA-dependent RNA polymerase are discussed.
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PMID:[Synthesis of oligoribonucleotides with the use of RNA polymerases of E. coli and immobilized synthetic DNA-templates]. 266 54

Site-directed in vitro mutagenesis was used to create analogs of human interferons (IFNs)-alpha 1 and -alpha 4. Analogs were expressed in vitro using SP6 RNA polymerase and a rabbit reticulocyte lysate cell-free protein synthesis system. Amino acid substitutions for the highly conserved residues at positions 33, 121, 122 and 123 greatly reduced the antiviral and antiproliferative activities on human cells of IFNs-alpha 1 and -alpha 4. In general, the amino acid substitutions had much less effect on the antiviral activities on bovine, compared with human, cells. Substitutions at positions 31, 41, 42, 124, 134, 135 and 136 had little or no effect on the biological activities of the IFN analogs. The abrogation of antiviral activity resulting from amino acid substitutions for the arginine residue at position 33 suggests that this arginine residue is required for binding to the IFN-alpha receptor on the cell surface.
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PMID:Functional significance of amino acid residues within conserved hydrophilic regions in human interferons-alpha. 268 50

A cDNA encoding the rat brain glucose transporter was inserted between the 5' and 3' untranslated regions from the Xenopus globin gene and downstream of an SP6 RNA polymerase start site. RNA synthesized from this vector was microinjected into oocytes from Xenopus laevis; this resulted in expression of the glucose transporter, as determined by both immunoblotting and the appearance of transport activity. The properties of the transporter were those expected from previous studies: it was glycosylated, and its activity, measured by 3-O-methylglucose transport, was inhibited by D-glucose and cytochalasin B, but not by L-glucose. The low level of endogenous glucose transport activity found in water-injected oocytes makes this a useful system in which to determine the kinetic parameters of transport. The Km for 3-O-methylglucose was found to be 20 mM under equilibrium exchange conditions. Despite the fact that oocytes exhibit insulin-dependent responses, insulin did not stimulate 3-O-methylglucose transport by injected oocytes.
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PMID:Expression of a functional glucose transporter in Xenopus oocytes. 269 9

We have studied the sequence requirements for 3'-end formation of rDNA transcripts in a cell-free system and show that the generation of correct ends of mouse pre-rRNA is brought about by a two-step process that involves a bona fide termination reaction, followed by a specific trimming of the primary transcript by 10 nucleotides. We show that termination of mouse ribosomal gene transcription by RNA polymerase I (pol I) takes place in front of an 18-bp DNA sequence element (the 'Sal box'), which was previously shown to function as termination signal. Termination of pol I transcription occurs at a fixed distance (11 bp) upstream of the Sal box, independent of the sequence of adjacent gene regions. The processing reaction, however, is strongly influenced by sequences flanking the termination signal at the 5' site. Substitution of a cluster of T residues by guanines within the region of 3'-end formation abolishes the 3'-terminal trimming of the primary transcript. Interestingly, this 3'-terminal processing event, which can be uncoupled from the termination reaction, requires both a correct 3' end and specific sequences in the 3'-terminal region of the primary transcript. Read-through transcripts generated in the extract system or by SP6 RNA polymerase are no substrate for the processing nuclease(s). Because the termination and processing activity can be separated chromatographically, the nucleolytic activity does not reside in TTF-I, the factor that binds to the Sal box and directs transcription termination.
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PMID:3'-end formation of mouse pre-rRNA involves both transcription termination and a specific processing reaction. 271 50


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