Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P50583 (asymmetrical)
 12,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The peripheral membrane protein fraction released by washing Acholeplasma laidlawii membranes with low-ionic strength buffers contained about 50% of the total membrane-bound ribonuclease and deoxyribonuclease activities. The ATPase, NADH oxidase and p-nitrophenylphosphatase activities remained bound to the membrane even when EDTA was added to the wash fluids, and thus appear to belong to the integral membrane protein group. Serving as a marker for peripheral membrane proteins, the membrane-bound ribonuclease activity was solubilized by bile salts much more effectively than the integral membrane-bound enzymes. On the other hand, the solubilized ribonuclease showed a much lower capacity to reaggregate with other solubilized membrane components to membranous structures. Yet, most of the ribonuclease molecules which were bound to the reaggregated membranes could not be released by low-ionic strength buffer. The reaggregated membranes differed from the native membranes in the absence of particles on their fracture faces obtained by freeze cleaving, and by their much higher labeling by the [125-I]lactoperoxidase iodination system. These results suggest that most of the proteins are exposed on the reaggregated membrane surfaces, with very little, if any, protein embedded in its lipid bilayer core. Enzyme disposition in the A. laidlawii membrane was studied by comparing the activity of isolated membranes with that of membranes of intact cells after treatment with pronase or with an antiserum to membranes. The data indicate the asymmetrical disposition of these activities, the ATPase and NADH oxidase being localized on the inner membrane surface, while the nucleases are exposed on the external membrane surface.
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PMID:Characterization of the mycoplasma membrane proteins. V. Release and localization of membrane-bound enzymes in Acholeplasma laidlawii. 23 52

The solution structure of human U1 snRNA was investigated by using base-specific chemical probes (dimethylsulfate, carbodiimide, diethylpyrocarbonate) and RNase V1. Chemical reagents were employed under various conditions of salt and temperature and allowed information at the Watson-Crick base-pairing positions to be obtained for 66% of the U1 snRNA bases. Double-stranded or stacked regions were examined with RNase V1. The dat gained from these experiments extend and support the previous 2D model for U1snRNA. However, to elucidate some aspects of the solution data that could not be accounted for by the secondary structure model, the information gathered from structure probing was used to provide the experimental basis required to construct and to test a tertiary structure model by computer graphics modeling. As a result, U1 snRNA is shown to adopt an asymmetrical X-shape that is formed by two helical domains, each one being generated by coaxial stacking of helices at the U1 snRNA cruciform. Chemical reactivities and model building show that a few nucleotides, previously proposed to be unpaired, can form A.G and U.U non Watson-Crick base-pairs, notably in stem-loop B. The structural model we propose for regions G12 to A124 integrates stereochemical constraints and is based both on solution structure data and sequence comparisons between U1 snRNAs.
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PMID:Solution structure of human U1 snRNA. Derivation of a possible three-dimensional model. 237 9

We have investigated in detail the secondary and tertiary structures of E. coli 16S rRNA binding site of protein S15 using a variety of enzymatic and chemical probes. RNase T1 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with 1-cyclohexyl-3 (2-(1-methylmorpholino)-ethyl)-carboiimide-p- toluenesulfonate (at U(N-3) and G(N-1)) and with diethylpyrocarbonate (at A(N-7)). The RNA region corresponding to nucleotides 652 to 753 was tested within: (1) the complete 16S rRNA molecule; (2) a 16S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S15-16S rRNA complex; (4) the S15-fragment complex. Cleavage and modification sites were detected by primer extension with reverse transcriptase. Our results show that: (1) The synthetized fragment folds into the same overall secondary structure as in the complete 16S rRNA, with the exception of the large asymmetrical internal loop (nucleotides 673-676/714-733) which is fully accessible in the fragment while it appears conformationally heterogeneous in the 16S rRNA; (2) the reactivity patterns of the S15-16S rRNA and S15-fragment complexes are identical; (3) the protein protects defined RNA regions, located in the large interior loop and in the 3'-end strand of helix [655-672]-[734-751]; (4) the protein also causes enhanced chemical reactivity and enzyme accessibility interpreted as resulting from a local conformational rearrangement, induced by S15 binding.
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PMID:The E. coli 16S rRNA binding site of ribosomal protein S15: higher-order structure in the absence and in the presence of the protein. 245 25

We report that pdxA, which is required for de novo biosynthesis of pyridoxine (vitamin B6) and pyridoxal phosphate, belongs to an unusual, multifunctional operon. The pdxA gene was cloned in the same 3.5-kilobase BamHI-EcoRI restriction fragment that contains ksgA, which encodes the 16S rRNA modification enzyme m6(2)A methyltransferase, and apaH, which encodes diadenosine tetraphosphatase (ApppA hydrolase). Previously, Blanchin-Roland et al. showed that ksgA and apaH form a complex operon (Mol. Gen. Genet. 205:515-522, 1986). The pdxA gene was located on recombinant plasmids by subcloning, complementation, and insertion mutagenesis, and chromosomal insertions at five positions upstream from ksgA inactivated pdxA function. DNA sequence analysis and minicell translation experiments demonstrated that pdxA encoded a 35.1-kilodalton polypeptide and that the stop codon of pdxA overlapped the start codon of ksgA by 2 nucleotides. The translational start codon of pdxA was tentatively assigned based on polypeptide size and on the presence of a unique sequence that was also found near the translational start of PdxB. This conserved sequence may play a role in translational control of certain pyridoxine biosynthetic genes. RNase T2 mapping of chromosomal transcripts confirmed that pdxA and ksgA were members of the same complex operon, yet about half of ksgA transcripts arose in vivo under some culture conditions from an internal promoter mapped near the end of pdxA. Transcript analysis further suggested that pdxA is not the first gene in the operon. These structural features support the idea that pyridoxine-biosynthetic genes are members of complex operons, perhaps to interweave coenzyme biosynthesis genetically with other metabolic processes. The results are also considered in terms of ksgA expression.
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PMID:Overlap between pdxA and ksgA in the complex pdxA-ksgA-apaG-apaH operon of Escherichia coli K-12. 267 Aug 94

bI1 RNA (excised from the first intron of the long form of the cytochrome b gene of Saccharomyces cerevisiae mitochondria) hybridizes with the two strands of a Bg/II-MboI DNA segment from this region. This fraction is resistant to digestions by DNase I and RNase T1 and disappears completely upon alkali hydrolysis. Strand-specific labeling of an intronic DNA fragment, cloned in pBR322 plasmid, was accomplished through the use of a T4 DNA polymerase. The purity of the probes was demonstrated by cloning an exon-intron fragment and labeling it by the same procedure; mRNA and pre-mRNA bands hybridized only with the transcribed DNA strand whereas bI1 RNA hybridized with the two strands under the stringent washing conditions employed (tm + 20 degrees C). Several experimental results argue against the possibility that the observation of two complementary bI1 RNA strands results from a partial self-complementarity of the RNA. A pre-mRNA intermediate from a box8 (G5046) mutant, still containing this intron, hybridizes only with the transcribed DNA strand of the pure intronic probe. The amount of the non-sense bI1 RNA strand is very low, in cells from two wild-type strains, relative to the sense RNA strand during the early stages of growth on glucose. It increases as the cells are released from glucose repression. bI1 RNA is resistant to RNase. Very little self-complementarity is seen by computer analysis of the sequence. Purified bI1 RNA is seen by electron microscopy under non-denaturing conditions as a mixture of double-stranded circular and linear molecules thus confirming the existence of the two complementary strands. The disappearance of all material following alkali hydrolysis demonstrates that these are indeed two RNA strands. Under fully denaturing conditions a mixture of single-stranded circular and linear molecules is seen as reported previously (Cell, 19, 321-329, 1980). We conclude that yeast mitochondria contain the two complementary bI1 RNA strands, one circular and the other linear. Considering a largely asymmetrical transcription of the mitochondrial genome in yeast and assuming that circularization of some intronic RNAs is part of RNA processing, we do not believe that the two strands are each a mixture of linear and circular molecules. The ratio of non-sense to sense bI1 RNA in a cytoplasmic petite mutant, A1B1, also varies according to growth conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Yeast mitochondria contain a linear RNA strand complementary to the circular intronic bI1 RNA of cytochrome b. 620 24

The substrate specificity of diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum for dinucleoside polyphosphates has been determined by high-performance liquid chromatography (HP-LC). Elution of a strong anion-exchange resin with a pH and ionic strength gradient of ammonium phosphate separates a series of monoadenosine and diadenosine polyphosphates. Most of the corresponding guanine nucleotides are also resolved on this HPLC system. One mole each of Ap4A and Gp4G is symmetrically hydrolyzed to 2 mol of ADP and GDP, respectively. Ap3A, Ap5A, Ap6A, and Ap4 are hydrolyzed, and in each case ADP is one of the products. Gp3G, Gp5G, Gp6G, and Gp4 are also substrates, and in each case GDP is one of the products. AMP, ADP, ATP, Ap2A, ADPR, GMP, GDP, GTP, NAD+, and NADP+ are not substrates. No hydrolysis of the cap dinucleotides m7Gp3Am and m7Gp3Cm was detected by HPLC. Diadenosine tetraphosphate pyrophosphohydrolase preparations were also assayed for adenylate kinase, nucleotide diphosphate kinase, NAD(P)+ pyrophosphohydrolase, phosphodiesterase, cyclic nucleotide phosphodiesterase, phosphatase, and ribonuclease activities. These enzymic activities were not detectable in diadenosine tetraphosphate pyrophosphohydrolase. The symmetrical hydrolysis of Ap4A and Gp4G is an unique catalytic property that distinguishes diadenosine tetraphosphate pyrophosphohydrolase from P. polycephalum from diadenosine tetraphosphate phosphohydrolases from other organisms.
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PMID:Diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase from Physarum polycephalum. Substrate specificity. 629 57

The interaction between human immunodeficiency virus type 1 (HIV-1) and RNA silencing pathways is complex and multifaceted. Essential for efficient viral transcription and supporting Tat-mediated transactivation of viral gene expression, the trans-activation responsive (TAR) element is a structured RNA located at the 5' end of all transcripts derived from HIV-1. Here, we report that this element is a source of microRNAs (miRNAs) in cultured HIV-1-infected cell lines and in HIV-1-infected human CD4+ T lymphocytes. Using primer extension and ribonuclease (RNase) protection assays, we delineated both strands of the TAR miRNA duplex deriving from a model HIV-1 transcript, namely miR-TAR-5p and miR-TAR-3p. In vitro RNase assays indicate that the lack of a free 3' extremity at the base of TAR may contribute to its low processing reactivity in vivo. Both miR-TAR-5p and miR-TAR-3p down-regulated TAR miRNA sensor activity in a process that required an integral miRNA-guided RNA silencing machinery. miR-TAR-3p exerted superior gene downregulatory effects, probably due to its preferential release from HIV-1 TAR RNA by the RNase III Dicer. Our study suggests that the TAR element of HIV-1 transcripts releases functionally competent miRNAs upon asymmetrical processing by Dicer, thereby providing novel insights into viral miRNA biogenesis.
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PMID:Identification of functional microRNAs released through asymmetrical processing of HIV-1 TAR element. 1829 84

The human genome contains more than 1,000 microRNA (miRNA) genes, which are transcribed mainly by RNA polymerase II. The canonical pathway of miRNA biogenesis includes the nuclear processing of primary transcripts (pri-miRNAs) by the ribonuclease Drosha and further cytoplasmic processing of pre-miRNAs by the ribonuclease Dicer. This review discusses the issue of miRNA end heterogeneity generated primarily by Drosha and Dicer cleavage and focuses on the structural aspects of the Dicer step of miRNA biogenesis. We examine the structures of miRNA precursors, both predicted and experimentally determined, as well as the influence of various motifs that disturb the regularity of pre-miRNA structure on Dicer cleavage specificity. We evaluate the structural determinants of the length diversity of miRNA generated by Dicer from different precursors and highlight the importance of asymmetrical motifs. Finally, we discuss the impact of Dicer protein partners on cleavage efficiency and specificity and propose the contribution of pre-miRNA structural plasticity to the dynamics of the dicing complex.
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PMID:The role of the precursor structure in the biogenesis of microRNA. 2160 69

Double-stranded RNAs consisting of 21-nucleotide passenger and guide strands, known as small interfering RNAs (siRNAs), can be used for the identification of gene functions and the regulation of genes involved in disease for therapeutics. The difficulty with unmodified siRNAs lies in the chemical synthesis of RNA, its degradation by RNase, the immune response derived from natural RNA, and the off-target effects mediated by the passenger strand. In this study, asymmetrical 18 base-paired double-strand oligonucleotides comprised of alternately combined DNAs and 2'-O-methyl RNAs, denoted as MED-siRNA, were evaluated. These modified oligonucleotides showed high RNase resistance, a reduced immune response, a highly efficient cleavage of target mRNA with binding to Argonaute 2 (Ago2) via RNA interference, and the subsequent reduction of target protein expression. These findings suggest the possibility of alternatives to unmodified siRNAs with potential use in therapeutics.
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PMID:Design of 2'-O-methyl RNA and DNA double-stranded oligonucleotides: naturally-occurring nucleotide components with strong RNA interference gene expression inhibitory activity. 3150 65