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)

A fully automated instrument for multiple simultaneous peptide synthesis was constructed to provide large numbers of peptides for immunological research. The synthesis is performed in a flow-through mode with the conventional solid supports contained in 48 individual reaction columns. The instrument is based on a commercial autosampler equipped with a motor-driven syringe for accurate delivery of reagents and a robot arm carrying a dispenser needle. Dedicated software was developed to compile overlapping peptides from a given protein sequence and to control all functions of the robot. In situ activation by BOP was chosen as the optimized chemistry protocol. The peptides are cleaved from the resin in the reactors used for synthesis, thus minimizing handling. Performance of the instrument was demonstrated by synthesis of overlapping 14-mer peptides derived from the sequence of HIV reversed transcriptase. A second mode of operation allows the synthesis to be carried out on the surface of polyethylene pins. Peptides derived from the sequence of human TNF were synthesized using this method and used to characterize antibodies raised against the intact protein.
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PMID:Automated multiple peptide synthesis. 128 42

Environmental and clinical isolates of mercury-resistant (resistant to inorganic mercury salts and organomercurials) bacteria have genes for the enzymes mercuric ion reductase and organomercurial lyase. These genes are often plasmid-encoded, although chromosomally encoded resistance determinants have been occasionally identified. Organomercurial lyase cleaves the C-Hg bond and releases Hg(II) in addition to the appropriate organic compound. Mercuric reductase reduces Hg(II) to Hg(O), which is nontoxic and volatilizes from the medium. Mercuric reductase is a FAD-containing oxidoreductase and requires NAD(P)H and thiol for in vitro activity. The crystal structure of mercuric ion reductase has been partially solved. The primary sequence and the three-dimensional structure of the mercuric reductase are significantly homologous to those of other flavin-containing oxidoreductases, e.g., glutathione reductase and lipoamide dehydrogenase. The active site sequences are the most conserved region among these flavin-containing enzymes. Genes encoding other functions have been identified on all mercury ion resistance determinants studied thus far. All mercury resistance genes are clustered into an operon. Hg(II) is transported into the cell by the products of one to three genes encoded on the resistance determinants. The expression of the operon is regulated and is inducible by Hg(II). In some systems, the operon is inducible by both Hg(II) and some organomercurials. In gram-negative bacteria, two regulatory genes (merR and merD) were identified. The (merR) regulatory gene is transcribed divergently from the other genes in gram-negative bacteria. The product of merR represses operon expression in the absence of the inducers and activates transcription in the presence of the inducers. The product of merD coregulates (modulates) the expression of the operon. Both merR and merD gene products bind to the same operator DNA. The primary sequence of the promoter for the polycistronic mer operon is not ideal for efficient transcription by the RNA polymerase. The -10 and -35 sequences are separated by 19 (gram-negative systems) or 20 (gram-positive systems) nucleotides, 2 or 3 nucleotides longer than the 17-nucleotide optimum distance for binding and efficient transcription by the Escherichia coli sigma 70-containing RNA polymerase. The binding site of MerR is not altered by the presence of Hg(II) (inducer). Experimental data suggest that the MerR-Hg(II) complex alters the local structure of the promoter region, facilitating initiation of transcription of the mer operon by the RNA polymerase. In gram-positive bacteria MerR also positively regulates expression of the mer operon in the presence of Hg(II).
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PMID:Bacterial resistances to inorganic mercury salts and organomercurials. 131 Nov 13

We constructed mercury resistance operon-luciferase (mer-lux) transcriptional fusion plasmids to evaluate in vivo gene expression rates of the mer structural gene promoter (PTPCAD) of transposon Tn21. In vivo gene expression kinetics corresponded well with those previously determined in vitro, yielding an apparent K0.5 for Hg(II)-stimulated induction by MerR of 9.3 x 10(-8) M with the same ultrasensitive threshold effect seen in vitro. We also used the mer-lux fusions to elucidate subtle variations in promoter activity brought about by altered superhelicity. Binding of inducer [Hg(II)] to the transcriptional activator MerR is known to result in DNA distortion and transcriptional activation of the mer operon; it has recently been demonstrated that this distortion is a consequence of MerR-Hg(II)-induced local DNA unwinding to facilitate RNA polymerase open complex formation at PTPCAD. Since negative supercoiling results in DNA unwinding similar to this MerR activation, we hypothesized that a global increase in plasmid supercoiling would facilitate MerR-mediated activation and compromise MerR-mediated repression, while removal of plasmid supercoils would compromise MerR's ability to induce transcription and facilitate its ability to repress transcription. Indeed, we found that increased negative supercoiling results in increased gene expression rates and decreased supercoiling results in reduced gene expression rates for the induced, repressed, and derepressed conditions of PTPCAD. Thus, luciferase transcriptional fusions can detect subtle variations in initial rates of gene expression in a real-time, nondestructive assay.
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PMID:A mer-lux transcriptional fusion for real-time examination of in vivo gene expression kinetics and promoter response to altered superhelicity. 133 70

Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond between the base and the ribose of the adenosine at position 4324 in eukaryotic 28 S rRNA. Ricin A-chain will also catalyze depurination in naked prokaryotic 16 S rRNA; the adenosine is at position 1014 in a GAGA tetraloop. The rRNA identity elements for recognition by ricin A-chain and for the catalysis of cleavage were examined using synthetic GAGA tetraloop oligoribonucleotides. The RNA designated wild-type, an oligoribonucleotide (19-mer) that approximates the structure of the ricin-sensitive site in 16 S rRNA, and a number of mutants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase. With the wild-type tetraloop oligoribonucleotide the ricin A-chain-catalyzed reaction has a Km of 5.7 microM and a Kcat of 0.01 min-1. The toxin alpha-sarcin, which cleaves the phosphodiester bond on the 3' side of G4325 in 28 S rRNA, does not recognize the tetraloop RNA, although alpha-sarcin does affect a larger synthetic oligoribonucleotide that has a 17-nucleotide loop with a GAGA sequence; thus, there is a clear divergence in the identity elements for the two toxins. Mutants were constructed with all of the possible transitions and transversions of each nucleotide in the GAGA tetraloop; none was recognized by ricin A-chain. Thus, there is an absolute requirement for the integrity of the GAGA sequence in the tetraloop. The helical stem of the tetraloop oligoribonucleotide can be reduced to three base-pairs, indeed, to two base-pairs if the temperature is decreased, without affecting recognition; the nature of these base-pairs does not influence recognition or catalysis by ricin A-chain. If the tetraloop is opened so as to form a GAGA-containing hexaloop, recognition by ricin A-chain is lost. This suggests that during the elongation cycle, a GAGA tetraloop either exists or is formed in the putative 17-member single-stranded region of the ricin domain in 28 S rRNA and this bears on the mechanism of protein synthesis.
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PMID:Ribosomal RNA identity elements for ricin A-chain recognition and catalysis. Analysis with tetraloop mutants. 137 5

We have developed two methods for selective 5' modification of RNAs generated by enzymatic synthesis using T7 RNA polymerase. The first method involves a two-step procedure. Transcription reactions are performed under standard conditions except that GTP is replaced by GTP gamma S. Since the polymerase initiates transcription with GTP, every transcript contains a 5' gamma-thiophosphate group which is modified with the thiol-specific reagent of choice (e.g., iodoacetyl dansyl derivative) in the second step. In an alternative method, transcription and modification reactions are carried out in a single step, using a mixture of dansylated GTP and GTP. Under the appropriate conditions, dansylated GTP effectively competes with GTP in the initiation reaction but does not substantially inhibit the elongation reaction. Yields of fluorescent 64-mer RNA ranging from 30 to 70% of the total transcription product have been obtained using these methods in combination with HPLC purification. This approach is amenable to large scale synthesis reactions and can be used to produce a wide variety of 5'-modified RNAs of virtually any size for structural or functional studies.
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PMID:Selective 5' modification of T7 RNA polymerase transcripts. 144 58

The gene product of braB encoding the Na+(Li+)-coupled carrier protein for L-leucine, L-isoleucine, and L-valine (LIV-II carrier) of Pseudomonas aeruginosa PML strain was identified and overexpressed using a T7 RNA polymerase/promoter plasmid system. The gene product was pulse-labeled with [35S]methionine as a protein of an apparent Mr of 34,000 on a sodium dodecyl sulfate-polyacrylamide gel. Cell membranes overproducing the LIV-II carrier were solubilized with n-dodecyl beta-D-maltopyranoside. The carrier protein was purified from the detergent extract by two purification steps: (i) immunoaffinity column chromatography using purified polyclonal antibody directed against synthetic 13-mer peptide corresponding to the carboxyl terminus region of the carrier and (ii) subsequent DEAE-cellulose column chromatography. The detergent was replaced by n-octyl beta-D-glucopyranoside prior to the first elution and phospholipid was present during purification. Proteoliposomes reconstituted with the purified LIV-II carrier exhibited Na+ or Li+ concentration gradient-driven transport of leucine, isoleucine, and valine. These results show that the LIV-II carrier was purified to be in a functional form.
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PMID:Immunoaffinity purification and reconstitution of sodium-coupled branched-chain amino acid carrier of Pseudomonas aeruginosa. 154 99

Homopyrimidine oligonucleotides bind to the major groove of a complementary homopyrimidine.homopurine stretch by triple helix formation. The bla gene from transposon Tn3 contains a homopyrimidine.homopurine sequence of 13 base pairs located just downstream of the RNA polymerase binding site. A 13-mer homopyrimidine oligonucleotide targeted to this sequence was tested for its effect on transcription of the bla gene in vitro. We show that the consequence of triple helix formation in front of the Escherichia coli RNA polymerase-promoter complex is to block the holoenzyme at its start site during a period that is dependent on temperature. The temperature dependence of transcription inhibition shows a direct correlation between this effect and the stabilization of the triple helix. Substitution of 5-methylcytosine to cytosine in the 13-mer oligonucleotide enhances triplex stability and transcription inhibition. Transcription inhibition by this synthetic repressor was also confirmed by footprinting studies demonstrating its specificity of action. The 13-mer oligonucleotide containing a psoralen derivative covalently linked to its 5' end shows an irreversible and specific inhibition of transcription initiation after exposure to light of wavelength greater than 310 nm.
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PMID:Specific inhibition of transcription by triple helix-forming oligonucleotides. 173 20

Philadelphia-chromosome positive chronic myeloid leukemia cells in chronic phase (CML-CP) or blast crisis (CML-BC) and normal bone marrow cells (NBMC) were incubated in vitro with antisense oligonucleotide specific against the BCR/ABL breakpoint junction to examine the possibility of selective inhibition of leukemia growth. Growth capability was determined in vitro by colony assay in semisolid medium in the presence of interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF). The 18-mer antisense directed against the specific BCR/ABL mRNA breakpoint region diminished the colony formation by CML-CP and CML-BC cells, but not by NBMC. Scrambled oligomer did not affect significantly the growth of leukemic and normal cells. If CML-BC cells were mixed with NMBC and incubated with specific BCR/ABL antisense oligomer, leukemic colonies were selectively inhibited, as was shown by reverse, transcriptase-polymerase chain reaction (RT-PCR) performed to detect BCR/ABL mRNA in single colonies. These results confirm the possibility of selective inhibition of leukemia cells by antisense treatment.
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PMID:Gene-targeted specific inhibition of chronic myeloid leukemia cell growth by BCR-ABL antisense oligodeoxynucleotides. 179 39

In this work we present the localization and characterization of the repl promoter (Prepl) and show aspects of the regulation. Comparison of Prepl with other autoregulated replication protein gene promoters revealed similarities, but Prepl differs from some of these characterized promoters in not being regulated by the heat-shock RNA polymerase. Primer extension analysis showed that Prepl is contained within five helically aligned 18 base pair repeats, or 18-mers of the previously defined minimal origin. In addition, we find that Prepl is autoregulated by a trans-acting product encoded in the REPI region. Purified Repl protein binds to the 18-mer region of the origin, suggesting that the repl gene is autoregulated by the protein product. The autoregulation appears to be co-operative since decreasing the 18-mer binding site region results in a concomitant non-linear loss of autorepression. The deletion derivatives show a decreased ability to bind the Repl protein when compared with origin DNA containing all of the binding region. The diminished capacity of the various deletion derivatives to bind Repl in vitro correlates with the loss of autorepression seen in vivo.
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PMID:Co-operative autoregulation of a replication protein gene. 180 40

The interaction between calf thymus topoisomerase II and DNA has been characterized using a transcription assay. A highly preferred recognition sequence for topoisomerase II was inserted in either direction downstream from a promoter specific for a bacteriophage RNA polymerase. The presence of topoisomerase II-DNA complexes on the template provoked blockage of transcription, yielding RNA transcripts terminated 5' to the topoisomerase II binding site. A footprint of topoisomerase II, derived from transcription towards the complex from either side, revealed that eukaryotic topoisomerase II binds a region of 28 base-pairs with a highly protected central core of 22 base-pairs. The binding region was located symmetrically around the topoisomerase II-mediated cleavage site. In agreement with this result, optimal topoisomerase II-mediated cleavage was observed with a DNA substrate consisting of a 28-mer oligonucleotide homologous to the protected region. Stepwise removal of base-pairs from the ends of the 28-mer gradually reduced the level of enzyme-mediated cleavage.
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PMID:Characterization of the interaction between topoisomerase II and DNA by transcriptional footprinting. 217 Jun 62


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