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)

Reverse transcriptase-like sequences (RTs) with amino acid motifs characteristic of non-LTR retrotransposons have been isolated from several medically important phlebotomine sandfly species. These sequences were amplified using the polymerase chain reaction (PCR) with primers based on conserved amino acid motifs present in previously described insect RTs. A further set of RTs were amplified using primers based on the conserved regions identified in phlebotomine RTs. The average similarity of the phlebotomine RTs to the Drosophila I, F and R1Dm elements was 28-29% between the closest primers used. Phlebotomine RTs were 31-62% similar to each other, the most dissimilar sequences coming from the same species. Several amino acid residues were invariant in the ten phlebotomine RTs, including motif Q(F/Y)GF, conserved in other non-LTR retrotransposons, but not in retrovirus or LTR retrotransposon RTs. The remarkable conservation of this distinctive domain of non-LTR retrotransposon RTs suggests it has a vital and possibly unique role in the mode of reverse transcription of this class of transposon.
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PMID:Isolation of non-LTR retrotransposon reverse transcriptase-like sequences from phlebotomine sandflies. 752 83

We have overproduced the leader peptidase from Escherichia coli in a high yield by using a T7 RNA polymerase/promoter system and purified the enzyme. This leader peptidase showed an apparent pH optimum of about 10 toward a synthetic peptide substrate, and was stable at temperatures below 40 degrees C. Kinetic studies indicated that one of the active site residues in the enzyme has a pKa value of approximately 7.5. The enzyme was rapidly inactivated by reaction with N-bromosuccinimide (NBS). When approximately two tryptophan residues were oxidized with NBS, the activity was almost completely lost and this inactivation was markedly prevented by a substrate. These NBS-reactive tryptophan residues were identified as Trp300 and Trp310 by a peptide mapping analysis. This indicates that Trp300 and/or Trp310 are critically important for the activity of the leader peptidase. On the other hand, the enzyme was scarcely inhibited by treatment with N-acetylimidazole, iodoacetic acid, 5,5'-dithiobis(2-nitrobenzoic acid), succinic anhydride, or 2,4,6-trinitrobenzenesulfonate. Diethylpyrocarbonate inhibited the enzyme; however, this inhibition did not seem to result from the modification of histidine residues. Thus, there seem to be no functionally important tyrosine, cysteine, or histidine residues or amino groups among the residues which readily react with these reagents. However, the enzyme was inactivated significantly by treatment with phenylglyoxal or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Therefore, some of the arginine residues and the carboxyl groups appear to be important for the enzyme activity.
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PMID:Leader peptidase from Escherichia coli: overexpression, characterization, and inactivation by modification of tryptophan residues 300 and 310 with N-bromosuccinimide. 762 19

We have reconstituted Bacillus subtilis trp attenuation in vitro. Purification of the mtrB gene product (TRAP) to near homogeneity allowed us to demonstrate that addition of this protein plus L-tryptophan to template, RNA polymerase, and nucleoside triphosphates caused transcription termination in the trpEDCFBA leader region. TRAP acts by binding to the nascent transcript and preventing formation of an RNA antiterminator structure, thereby allowing terminator formation and transcription termination. Oligonucleotides complementary to segments of the antiterminator were used to demonstrate that formation of this RNA hairpin was responsible for transcription read-through. TRAP was found to be a 60-kDa multimeric protein composed of identical 6- to 8-kDa subunits, and its elution profile on a chromatographic column did not change in the presence of tryptophan.
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PMID:Reconstitution of Bacillus subtilis trp attenuation in vitro with TRAP, the trp RNA-binding attenuation protein. 767 34

The genomes of Lilium species are very large, containing 30-40 million kilobase pairs of DNA. An abundant fragment of 3.5 kb was released by BamHI digestion of genomic DNA of Lilium speciosum. Analysis of 20 genomic clones containing sequences homologous to the fragment showed it to be part of a 4.45 kb dispersed repeat, which was named del2. Sequence analysis of one full element and regions of four others revealed del2 to be a non-LTR (long terminal repeat) retrotransposon. It is flanked by short direct repeats of from 4 to 13 bp and a run of adenines occurs at one end (the proposed 3' end), 63 bp downstream from a polyadenylation signal. A possible RNA polymerase II promoter similar to that found in Drosophila I and F group elements is present internally 30 bp downstream from the 5' end. Two degenerate open reading frames (ORFs) are present, the 5' ORF containing a gag-related cysteine motif, and the 3' ORF containing a different cysteine motif also found in most non-LTR retrotransposons. The 3' ORF also has regions with homology to reverse transcriptase sequences, which are most similar to those in Cin4 of maize, the L1 LINE elements of humans and mice and the R2 ribosomal DNA inserts of insects. The majority of del2 elements occur as the full 4.45 kb element. They account for an estimated 4% of the L. speciosum genome and are present in approximately 250,000 copies. del2-related sequences were also detected in 12 other monocot species.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:An abundant LINE-like element amplified in the genome of Lilium speciosum. 768 Nov 39

Termination of mouse ribosomal gene transcription by RNA polymerase I (Pol I) requires the specific interaction of a DNA binding protein, mTTF-I, with an 18 bp sequence element located downstream of the rRNA coding region. Here we describe the molecular cloning and functional characterization of the cDNA encoding this transcription termination factor. Recombinant mTTF-I binds specifically to the murine terminator elements and terminates Pol I transcription in a reconstituted in vitro system. Deletion analysis has defined a modular structure of mTTF-I comprising a dispensable N-terminal half, a large C-terminal DNA binding region and an internal domain which is required for transcription termination. Significantly, the C-terminal region of mTTF-I reveals striking homology to the DNA binding domains of the proto-oncogene c-Myb and the yeast transcription factor Reb1p. Site-directed mutagenesis of one of the tryptophan residues that is conserved in the homology region of c-Myb, Reb1p and mTTF-I abolishes specific DNA binding, a finding which underscores the functional relevance of these residues in DNA-protein interactions.
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PMID:Different domains of the murine RNA polymerase I-specific termination factor mTTF-I serve distinct functions in transcription termination. 772 Jul 15

On amino acid starvation, Escherichia coli cells exhibit an adaptive facility termed the stringent response. This is characterized by the production of high levels of a regulatory nucleotide, ppGpp, and concomitant curtailment in rRNA synthesis. Various studies reported earlier indicated that RNA polymerase is the site of action of ppGpp although a direct demonstration of the interaction of ppGpp with E. coli RNA polymerase is still lacking. Here we report the labelling of ppGpp with a fluorescent probe, 1-aminonapthalene-5-sulphonate (AmNS), at the terminal phosphates. AmNS-ppGpp responded much like a ppGpp molecule in an in vitro total transcription assay at selective promoters. Fluorescence titration of the tryptophan emission of RNA polymerase by AmNS-ppGpp indicated a unique binding site in the absence of template DNA. Competition experiments showed that unlabelled ppGpp binds to the enzyme at the same site. Sigma factor seems to have no effect on this binding. The titration profile is also characterized by a single slope in the Scatchard analysis. The presence of GTP or GDP does not influence the binding of AmNS-ppGpp with RNA polymerase. Forster's distance measurement was carried out which placed AmNS-ppGpp 27 A away from the rifampicin-binding domain of RNA polymerase.
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PMID:Evidence for a ppGpp-binding site on Escherichia coli RNA polymerase: proximity relationship with the rifampicin-binding domain. 774 47

To study the contributions of the hemagglutinin-neuraminidase (HN) and the fusion (F) glycoproteins in virus-induced membrane fusion, the HN and F proteins of human parainfluenza virus type-1 (hPIV-1) and Sendai virus (SV) were expressed in HeLa T4+ cells using the vaccinia virus-T7 RNA polymerase transient expression system. Expression of F protein alone did not induce cell fusion. However, coexpression of homologous F and HN proteins resulted in extensive syncytium formation by hPIV-1 or SV glycoproteins, which supports the proposal that both the F and HN glycoproteins are necessary for membrane fusion. To investigate the function of HN in membrane fusion, we coexpressed heterologous combinations of the HN and F proteins of hPIV-1 and SV. No fusion was observed when SV HN and hPIV-1 F proteins were coexpressed. In contrast, the coexpression of hPIV-1 HN and SV F induced extensive cell fusion. These results suggest that specific interaction between HN and F is required to induce membrane fusion. To locate regions that are essential to the fusion promoting activity, chimeric HN proteins of SV and hPIV-1 were constructed. The chimeric proteins coexpressed with the SV or hPIV-1 F proteins indicated that some regions in the middle 62% of HN contribute to the fusion-promoting activity. To determine the role of the transmembrane region of HN on fusion-promoting activity, mutant HN proteins were expressed and their biological activities examined. Mutation of hPIV-1 HN at residue 55 from cysteine to tryptophan did not affect cell binding, neuraminidase activities, or homooligomer formation, but did result in the loss of cell fusion activity. The mutation of the same cysteine residue to glycine retained the fusion-promoting activity, suggesting that a sulfhydryl moiety is not specifically required at position 55, but the structure of the residue that occupies the position is important in fusion-promoting activity.
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PMID:Regions on the hemagglutinin-neuraminidase proteins of human parainfluenza virus type-1 and Sendai virus important for membrane fusion. 794 17

A simple fluorimetric assay based on internal fluorescence of tryptophan residues of E. Coli RNA polymerase has been developed to ascertain the number of steps during conversion of closed complex of the polymerase-promoter (trp promoter cloned in plasmid pDR720) to open complex. Our results from measurement on relative ratio of fluorescence at 340 nm (lambda ex = 295 nm) for free and promoter-bound RNA polymerase as a function of temperature, within the range 4 degrees C to 37 degrees C, indicate following equilibria for the above conversion: R+P<-->RPc<-->RPi1<-->RPi2<-->RPo. Apart from detection of one more intermediate in terms of conformational states of the bound RNA polymerase, second feature of our studies is the examination of conformational state of the polymerase using accessibility of fluorophor, tryptophan residues, to a neutral quencher, acrylamide, as the probe. We observe that in terms of accessibility of tryptophan residues in protein, intermediate complex, RPi2, is conformationally most perturbed in comparison to free polymerase. Implications of these results are discussed and compared with the available reports from footprinting and gel retardation assays of RNA polymerase-promoter interactions.
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PMID:Intrinsic fluorescence of E. coli RNA polymerase as a probe for its conformational changes during transcription initiation. 800 19

Region 2 of eubacterial sigma factors is highly conserved and the subdomain 2.4 is involved in -10 promoter recognition. An evolutionary conserved "RpoD box" has been identified at the junction of subdomain 2.3/2.4 in class I and class II sigma factors and there are two tryptophan residues at position 433 and 434 which can be used as intrinsic fluorescent markers to study their structure-function relationship. Site-directed mutagenesis of these two tryptophan residues has been carried out to generate three variants of sigma 70 of Escherichia coli RNA polymerase. These are W433F, W433G and W434G. sigma 70-W433F is found to be indistinguishable from the native sigma factor by both structural and functional analysis. sigma 70-W433G shows anomalous mobility on SDS-PAGE like the native sigma factor, is alpha-helical in conformation (50% helicity) although found to be less active in total transcription when reconstituted with core RNA polymerase. Free sigma 70-W434G, unlike the native sigma factor, shows the expected mobility of a 70 kDa protein on SDS-PAGE and has 20% helicity. Time-resolved fluorescence analysis indicates that free sigma 70-W434G has DNA binding ability, and displays a normal abortive initiation reaction but a decreased level of productive transcription after reconstitution with core RNA polymerase. A model is proposed in which tryptophan at position 434 interacts with the hydrophobic 1.1 domain of sigma 70 giving rise to the stability of the protein under denaturing conditions.
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PMID:A point mutation at the junction of domain 2.3/2.4 of transcription factor sigma 70 abrogates productive transcription and restores its expected mobility on a denaturing gel. 807 73

This study was designed to clarify the important association between eosinophilia-myalgia syndrome (EMS) and the L-tryptophan contaminant, "Peak E." To determine the functional activation of eosinophils induced by Peak E, eosinophil cationic protein (ECP) release was examined. Peak E augumented the release of ECP from peripheral blood normodense eosinophils by degranulation. Proliferative analysis using the human eosinophilic leukemia cell line EoL-3 showed prominent cellular replication in the presence of Peak E. Moreover, Peak E upregulated interleukin 5 (IL-5) receptor levels on normodense eosinophils. Of particular interest, Peak E-stimulated human splenic T cells produced bioactive and immunoreactive IL-5. Marked induction of IL-5 mRNA in Peak E-stimulated T cells was also shown by reverse-transcriptase polymerase chain reaction (RT-PCR). In contrast, L-tryptophan without the contaminant showed none of these effects. Thus, these data suggest that Peak E might be involved in the pathogenesis of EMS through bimodal mechanism including IL-5 generation by T cells and potentiation of eosinophil functional activation.
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PMID:1,1'-Ethylidenebis(tryptophan) (Peak E) induces functional activation of human eosinophils and interleukin 5 production from T lymphocytes: association of eosinophilia-myalgia syndrome with a L-tryptophan contaminant. 813 37


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