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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)

Nucleosome formation on inverted repeats or on some alternations of purines and pyrimidines can be inhibited in vitro by DNA supercoiling through their supercoiling-induced structural transitions to cruciforms or Z-form DNA, respectively. We report here, as a result of study of single nucleosome reconstitutions on a DNA minicircle, that a physiological level of DNA supercoiling can also enhance nucleosome sequence preference. The 357 base-pair minicircle was composed of a promoter of phage SP6 RNA polymerase joined to a 256 base-pair fragment containing a sea urchin 5 S RNA gene. Nucleosome formation on the promoter was found to be enhanced on a topoisomer with in vivo superhelix density when compared to topoisomers of lower or higher superhelical densities, to the nicked circle, or to the linear DNA. In contrast, nucleosomes at other positions appeared to be insensitive to supercoiling. This observation relied on a novel procedure for the investigation of nucleosome positioning. The reconstituted circular chromatin was first linearized using a restriction endonuclease, and the linear chromatin so obtained was electrophoresed as nucleoprotein in a polyacrylamide gel. The gel showed well-fractionated bands whose mobilities were a V-like function of nucleosome positions, with the nucleosome near the middle migrating less. This behavior is similar to that previously observed for complexes of sequence-specific DNA-bending proteins with circularly permuted DNA fragments, and presumably reflects the change in the direction of the DNA axis between the entrance and the exit of the particle. Possible mechanisms for such supercoiling-induced modulation of nucleosome formation are discussed in the light of the supercoiling-dependent susceptibility to cleavage of the naked minicircle with S1 and Bal31 nucleases; and a comparison between DNase I cleavage patterns of the modulated nucleosome and of another, non-modulated, overlapping nucleosome.
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PMID:Chromatin reconstitution on small DNA rings. IV. DNA supercoiling and nucleosome sequence preference. 131 7

Adducts produced by modification of DNA with benzo[a]pyrene diolepoxide (BPDE) are known to inhibit both DNA and RNA synthesis. This phenomenon has been used as a method for determining the distribution of carcinogen binding within defined DNA sequences. A critical comparison of different enzyme activities on adducted DNA is needed, since different enzymes may process adducted DNA differently. Thus, we compared blocks in DNA polymerase activity with that of an RNA polymerase and with an exonuclease at single base resolution. BPDE adducts blocked the progression of cloned T7 DNA polymerase (Sequenase) in a dose-dependent manner. Although the majority of these blocks were at one base prior to adducted guanines, we also observed some blocks opposite specific guanines, suggesting that in some sequences the polymerase inserted a base opposite the modified guanine. Digestion with T4 DNA polymerase (3'----5') exonuclease activity was also blocked in BPDE-adducted DNA; however, fragments produced by blocks in T4 exonuclease migrated two or more bases longer than the corresponding guanine. Mapping of adduct distributions using both Sequenase and T4 exonuclease gave similar results, demonstrating that a long tract of guanines was preferentially modified, and within a polyguanine sequence, the 5' guanines were more heavily modified than the 3' guanines. Transcription of adducted DNA by SP6 RNA polymerase was also inhibited in a dose-dependent manner. However, adducted bases which posed strong blocks to the DNA polymerase were not always strong blocks to the RNA polymerase. Thus, in terms of adduct distribution, Sequenase and T4 exonuclease provided more consistent results than the RNA polymerase, since blockage of the RNA polymerase correlated poorly with guanines.
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PMID:DNA polymerase, RNA polymerase and exonuclease activities on a DNA sequence modified by benzo[a]pyrene diolepoxide. 132 70

Full-length and 5'-truncated variants of human (h) tRNA(UUULys3) were synthesized by in vitro transcription using SP6 RNA polymerase. Bovine(b) tRNA(SUULys3) was purified from calf liver. Both full-length tRNA species were shown to be biologically active in an aminoacylation assay. Gel retardation assays revealed that both full-length tRNA species, as well as a 5'-truncated h-tRNA(UUULys3) molecule containing 24 nucleotides (nt) at the 3' end (Lys24), interact with human immunodeficiency virus (HIV)-1 reverse transcriptase (RT). Competition studies with these three tRNA species demonstrate that the 3' end of h-tRNA(UUULys3) contributes to the interaction with HIV-1 RT. Escherichia coli tRNA(UUULys) and tRNA(UUCGlu2) were also able to interact with the enzyme, whereas unrelated RNA molecules such as E. coli 5S rRNA did not bind to RT. Both b-tRNA(SUULys3) and h-tRNA(UUULys3) molecules, as well as the 5'-truncated variants, could be demonstrated to prime cDNA synthesis specifically using a HIV-1 RNA template, prepared by in vitro transcription, indicating that other viral or cellular proteins are not essential for this process. E. coli tRNA(UUULys) and tRNA(UUCGlu2), although able to interact with HIV-1 RT, failed to prime retroviral transcription. Products of cDNA synthesis were characterized by polymerase chain reaction, demonstrating that at least 18 nt at the 3' ends of h-tRNA(UUULys3) and b-tRNA(SUULys3) are still present in the cDNA product, whereas the 5' ends of both primer molecules were removed by the RNase H activity of HIV-1 RT.
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PMID:Synthetic human tRNA(UUULys3) and natural bovine tRNA(UUULys3) interact with HIV-1 reverse transcriptase and serve as specific primers for retroviral cDNA synthesis. 137 59

We have determined the fate of a nucleosome core on transcription. A nucleosome core was assembled on a short DNA fragment and ligated into a plasmid containing a promoter and terminators for SP6 RNA polymerase. The nucleosome core was stable in the absence of transcription. The distribution of nucleosome cores after transcription was examined. The histone octamer was displaced from its original site and reformed a nucleosome core at a new site within the same plasmid molecule, with some preference for the untranscribed region behind the promoter. These observations eliminate several models that have been proposed for transcription through a nucleosome core. Our results suggest that a nucleosome core in the path of a transcribing polymerase is displaced by transfer to the closest acceptor DNA.
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PMID:A nucleosome core is transferred out of the path of a transcribing polymerase. 139 27

We have previously isolated and sequenced Nicotiana cytoplasmic tRNA(Tyr) with G psi A anticodon which promotes readthrough over the leaky UAG termination codon at the end of the 126 K cistron of tobacco mosaic virus RNA and we have demonstrated that tRNA(Tyr) with Q psi A anticodon is no UAG suppressor. Here we show that the nucleotide in the middle of the anticodon (i.e., psi 35) also contributes to the suppressor efficiency displayed by cytoplasmic tRNA(Tyr). A tRNA(Tyr) with GUA anticodon was synthesized in vitro using T7 RNA polymerase transcription. This tRNA(Tyr) was unable to suppress the UAG codon, indicating that nucleotide modifications in the anticodon of tRNA(Tyr) have either stimulating (i.e., psi 35) or inhibitory (i.e., Q34) effects on suppressor activity. Furthermore, we have shown that the UAA but not the UGA stop codon is also efficiently recognized by tobacco tRNA(G psi ATyr), if placed in the TMV context. Hence this is the first naturally occurring tRNA for which UAA suppressor activity has been demonstrated. In order to study the influence of neighbouring nucleotides on the readthrough capacity of tRNA(Tyr), we have established a system, in which part of the sequence around the leaky UAG codon of TMV RNA was inserted into a zein pseudogene which naturally harbours an UAG codon in the middle of the gene. The construct was cloned into the vector pSP65 and in vitro transcripts, generated by SP6 RNA polymerase, were translated in a wheat germ extract depleted of endogenous mRNAs and tRNAs. A number of mutations in the codons flanking the UAG were introduced by site-directed mutagenesis. It was found that changes at specific positions of the two downstream codons completely abolished the readthrough over the UAG by Nicotiana tRNA(Tyr), indicating that this tRNA needs a very specific codon context for its suppressor activity.
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PMID:Pseudouridine in the anticodon G psi A of plant cytoplasmic tRNA(Tyr) is required for UAG and UAA suppression in the TMV-specific context. 146 24

We have compared RNA polymerase promoter activities in PCR-generated DNA fragments for use in the in vitro transcription of cRNA probes. Sense oligonucleotide primers, specific for the mouse acidic fibroblast growth factor gene, were synthesized with 5' extensions containing promoter sequences for the T7, T3 and SP6 RNA polymerase promoters. A common antisense primer was used with each of the promoter/aFGF primers to prepare PCR-generated DNA fragments (minigenes). In vitro transcription efficiency for each of these constructs was evaluated by incorporation of radioactivity into the cRNA products. We find that both the T7 and T3 promoters can direct the synthesis of cRNA probes of high specific activity from a PCR-generated DNA fragment, but that SP6 cannot. No detectable cRNA product was obtained using either T7 polymerase on the T3/minigene or T3 on the T7/minigene. Antisense cRNA probes, transcribed from minigene constructs were used for both Northern and in situ hybridization studies. A PCR-generated DNA fragment with RNA polymerase promoter sequences at each end provides a single template for synthesis in vitro of either sense or antisense cRNA probes.
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PMID:Synthesis of cRNA probes from PCR-generated DNA. 147 30

A shuttle cosmid vector has been constructed for Escherichia coli and actinomycetes. This vector, pFD666, utilizes the origin of replication (ori) of the broad-host-range plasmid, pJV1, from Streptomyces phaeochromogenes, for replication in actinomycetes and is compatible with vectors derived from pIJ101. The pFD666 vector employs the neomycin phosphotransferase-encoding gene (neo) from transposon Tn5 as the selective marker. To achieve this, the native promoter of neo was replaced by one optimized for expression in both hosts. The polylinker used for cloning has nine unique sites flanked by the promoters for T7 and SP6 RNA polymerase for the production of specific RNA probes. Terminators on both sides of the polylinker protect the vector from transcription originating from cloned inserts. An M13 ori allows the production of single-stranded DNA.
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PMID:A versatile shuttle cosmid vector for use in Escherichia coli and actinomycetes. 154 47

Ricin B chain is an N-glycosylated galactose-specific lectin. Examination of the amino acid sequence of the protein has shown it to be the product of a series of gene duplication events based on an original galactose binding peptide. The X-ray crystallographic structure of the protein reveals that it consists of two globular domains, each composed of three smaller subdomains. In each globular domain only one of the three subdomains has retained its ability to bind galactose. Through DNA manipulation we have created a series of fusions of portions of ricin B chain, each carrying only one galactose binding site, to the ricin signal sequence. Transcripts synthesized in vitro using SP6 RNA polymerase were injected into Xenopus oocytes where the recombinant proteins were produced in a mature form. The products were shown to be N-glycosylated and produced in a soluble stable form. Also, they retained the ability to bind galactose. Preliminary experiments on the reassociation of these ricin B chain fragments with ricin A chain to create a modified holotoxin were also carried out.
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PMID:Recombinant ricin B chain fragments containing a single galactose binding site retain lectin activity. 155 Mar 53

The cleavage signal-1 protein (CS-1), a doublet antigen comprised of approx. 14-kDa and 18-kDa proteins has been shown to be present on the surface of sperm of various mammalian species including humans. Polyclonal antibodies to CS-1 inhibit the early cleavage of fertilized eggs without apparently affecting sperm penetration and pronuclear formation. We report here the cloning of the human CS-1 cDNA and its expression in vitro to obtain the recombinant protein (reCS-1) molecule. The CS-1 cDNA clone was isolated by immunological screening of a human testis lambda gt11 cDNA library with mono-specific polyclonal antibody against CS-1. The cDNA is 1828 bp long; the start codon assigned to the first ATG (bp 98-100) encodes a protein with 249 amino acid residues terminating at TAA (bp 845-847). The cDNA isolated has a 97-bp 5' and a 984-bp 3' untranslated region. The potential polyadenylation signal (5'-AATAAA) is at bp 1803-1808. An extensive computer search of the GenBank database did not indicate any extensive homology with any known sequence, indicating that CS-1 is a unique protein. The CS-1 cDNA was cloned in the transcription vector, pGEM-11Zf, to obtain high-level in vitro transcription by SP6 and T7 RNA polymerase. The transcribed CS-1 RNA was translated in a rabbit reticulocyte in vitro translation system and produced a 33-kDa reCS-1 protein, as assessed by migration in a SDS-polyacrylamide gel.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Human cleavage signal-1 protein; cDNA cloning, transcription and immunological analysis. 155 70

The phage T7 and SP6 RNA polymerase-promoter systems are very similar in many characteristics, but maintains stringent specificity for each. In order to identify the base pair element that distinguishes between T7 and SP6 promoters, the base pairs at -12, -10, -9, and -8 of the T7 promoter consensus sequence were changed singly and multiply to the SP6 promoter-specific base pairs, and assayed for T7 and SP6 promoter activities. The results indicate that the primary discrimination element is the base pairs at -8 and -9. The two-base-pair substitution alone in T7 promoter by SP6-specific base pairs is sufficient to make the T7 variant be a SP6 promoter, abolishing T7 promoter activity.
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PMID:A two-base-pair substitution in T7 promoter by SP6 promoter-specific base pairs alone abolishes T7 promoter activity but reveals SP6 promoter activity. 161 86


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