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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Specific binding of the human immunodeficiency virus Tat protein to its RNA site (TAR) is mediated largely by a single arginine residue located within a basic region of the protein. Many essential features of the interaction can be mimicked by the free amino acid arginine, and an NMR model has been proposed in which the arginine guanidinium group binds to a guanine base in the major groove and to two phosphates adjacent to a bulge, with the RNA structure stabilized by a base triple between a U in the bulge and an adjacent A:U base pair. To compare the TAR structure to other arginine-binding RNAs, we performed in vitro selection experiments and identified RNAs with arginine-binding affinities similar to TAR. About 40% of the selected RNAs contained the same motif found in TAR: two stems separated by a bulge of at least two nucleotides, a U at the 5' position of the bulge, and G:C and A:U base pairs above the bulge. In many cases, the upper stems contained only the G:C and A:U pairs, located next to small loops. Chemical modification experiments demonstrated that these "TAR-like" RNAs bound arginine in a manner similar to TAR, and in some cases identified nucleotides outside the binding site that contributed to binding. To explore how small loops might help stabilize the structures of adjacent arginine-binding sites, we measured arginine-binding affinities of TAR-like RNAs having all possible three-nucleotide loops. An RNA with a UAG loop bound with highest affinity, and chemical modification and RNase mapping experiments suggested that the RNA changes conformation upon arginine binding, converting a large unstructured loop into a bulge conformation related to that of TAR. The results suggest that the arginine-binding site in TAR is structurally versatile and demonstrate how binding can be modulated by the surrounding RNA context.
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PMID:Arginine-binding RNAs resembling TAR identified by in vitro selection. 865 64

To examine the role of polyadenylation in the nuclear export of mRNA, we have replaced the poly(A) signal in a Rev-responsive human immunodeficiency virus type 1-based reporter gene with a cis-acting hammerhead ribozyme. Transcripts from this gene thus acquire a 3' terminus by cis-ribozyme cleavage rather than by polyadenylation. The nuclear and cytoplasmic distribution of transcripts was investigated using transient gene expression and quantitative RNase protection assays. In the absence of Rev, a basal level of polyadenylated unspliced mRNA transcribed from a poly(A) signal-containing control reporter gene was detected in the cytoplasm of transfected COS7 cells. However, cytoplasmic ribozyme-cleaved unspliced RNA was only barely detectable. The nuclear/cytoplasmic (n/c) ratio of polyadenylated RNAs was 3.8, while the n/c ratio for ribozyme cis-cleaved RNAs was 33. The cytoplasmic localization of the polyadenylated unspliced mRNA was enhanced about 10-fold in the presence of Rev and the Rev-responsive element. In marked contrast to this, ribozyme cleaved RNA accumulated almost exclusively (n/c ratio of 28) in the nucleus in the presence of Rev. Actinomycin D time course analysis suggested that the low levels of the cytoplasmic ribozyme-cleaved RNAs in both the presence and absence of Rev were due to serve export deficiency of ribozyme-cleaved RNA. Finally, by inserting a 90-nucleotide poly(A) stretch directly upstream of the ribozyme cassette, we have demonstrated that a long stretch of poly(A) near the 3' end of a ribozyme-cleaved transcript is not sufficient for directing mRNA export. Taken together, these results suggest that polyadenylation is required for the nucleocytoplasmic transport of mRNA and that Rev interaction with the Rev-responsive element cannot bypass this requirement.
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PMID:Role of polyadenylation in nucleocytoplasmic transport of mRNA. 865 27

A target RNA/DNA-specific nuclease could be constructed if a specific RNA/DNA binding domain allowing target RNA/DNA recognition was fused to a (deoxy)ribonucleolytic domain allowing target RNA/ DNA cleavage. The design and construction of such a chimeric enzyme could be of value for both basic research involving structure-function relationships and applied research requiring inactivation of harmful RNA/DNA molecules of cellular or pathogenic origin. The feasibility of this designer nuclease approach for inactivating specific RNA/DNA molecules was assessed using human immunodeficiency virus type-1 (HIV-1) RNA as a model. Trans-activator of transcription (Tat) protein is one of the key regulatory proteins encoded by HIV-1. It binds to the trans-activation-responsive (TAR) RNA element located within the 5' non-coding region of HIV-1 RNAs. The TAR RNA binding domain of this protein was fused to the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT). RNase H by itself lacks an RNA binding domain. The chimeric Tat-RNase H protein was shown to specifically recognize and cleave HIV-1 TAR RNA in vitro. Cleavage was abolished by mutations in the Tat binding region within the TAR RNA, indicating that it is specific to HIV-1 TAR RNA.
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PMID:Fusion with an RNA binding domain to confer target RNA specificity to an RNase: design and engineering of Tat-RNase H that specifically recognizes and cleaves HIV-1 RNA in vitro. 865 73

The RNase H family of enzymes catalyzes the hydrolysis of RNA from RNA DNA hybrids in a divalent metal-dependent fashion. To date, structure/function studies have focused on two members of this family: Escherichia coli RNase HI, a small monomeric protein; and human immunodeficiency virus, type I (HIV) RNase H, a domain of HIV reverse transcriptase. The isolated RNase H domain from HIV reverse transcriptase can be expressed independently and shares significant structural homology with its E. coli homologue; however, unlike the bacterial protein, it is inactive. The most notable difference between the inactive domain from HIV and the active E. coli protein is a basic helix/loop sequence, present in E. coli but absent from the HIV homologue. Substitution of this basic region into the HIV domain partially restores its activity and increases its thermodynamic stability. By deleting the basic helix/loop region, we have modeled the structural difference between these two polypeptides onto the E. coli homologue. Surprisingly, the resulting mutant protein is active in Mn2+-dependent fashion. Therefore, the basic helix/loop is not required for RNase H activity.
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PMID:The putative substrate recognition loop of Escherichia coli ribonuclease H is not essential for activity. 870

Ribonucleases appear to have physiologic roles in host defense against cancer, viruses, and other parasites. Previously it was shown that select ribonucleases added to cells concurrently with virions blocked human immunodeficiency virus, type I (HIV-1) infection of H9 cells. We now report that a ribonuclease homologous to RNase A, named onconase, inhibits virus replication in chronically HIV-1-infected human cells without killing the virally infected cell. Examining the mechanism of this inhibition shows that onconase enters the infected cells and degrades HIV-1 RNA without degrading ribosomal RNA or the three different cellular messenger RNAs analyzed. The homologous human pancreatic RNase lacks anti-viral activity. Comparing recombinant forms of onconase and a onconase-human RNase chimera shows that the N-terminal 9 amino acids and the pyroglutamyl residue of onconase are required for full anti-viral activity. Thus extracellular ribonucleases can enter cells, metabolize select RNAs, and inhibit HIV virion production within viable replicating cells.
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PMID:Inhibition of HIV-1 production and selective degradation of viral RNA by an amphibian ribonuclease. 870 32

Unique transcriptional transactivation by the human immunodeficiency virus type 1 (HIV-1) Tat protein of long terminal repeat (LTR)-driven RNA expression, in the absence of the transactivator responsive element (TAR), was previously demonstrated in central nervous system (CNS)-derived astrocytic cell-lines, including U87MG. In the present study, RNase protection assays were utilized to reveal the molecular mechanism(s) underlying transactivation of the HIV-1-LTR in these cells. Short transcripts, which represent abortive HIV-1 transcription, could not be detected either in the absence or presence of Tat, and no differences in transcript levels were detected using 5' probes, as compared to 3' probes, in the experiments. Thus, the transactivational effects of Tat, in U87MG cells, were potentially based on the increase of transcriptional initiation, both in TAR-dependent and -independent states. Further, by using newly established stable cellular transformant, containing HIV-1-LTR-reporter gene constructs, TAR-independent transactivation was demonstrated to efficiently function primarily in transiently-transfected U87MG cells. U87MG cells, stably-transfected with the intact HIV-1 proviral genome, produced very low levels of virus after long-term culture, as previously reported in other astrocytic cells. These cells demonstrated profoundly restricted transcription of the HIV-1 genome, with no detectable levels of HIV-1-specific RNA by Northern blotting, indicating that the restriction of viral production in these cells is principally due to the low level of overall transcription from the 5' HIV-1-LTR. Transcription of HIV-1 RNA in this cell could not be significantly up-regulated by various stimulators, such as phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor-alpha (TNF-alpha) and sodium butyrate. These data suggest that the restriction of HIV-1 transcription in these cells may be controlled by different mechanism(s) from those in lymphocytic or monocytic cells.
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PMID:Mechanisms of transcriptional transactivation and restriction of human immunodeficiency virus type I replication in an astrocytic glial cell. 871 Mar 70

The amphiphilic novenamines described in this report have been shown previously to be specific inhibitors of human immunodeficiency virus type 1 reverse transcriptase-associated ribonuclease, which they inhibit when they are in the micellar state but not when they are monomeric. These compounds also inhibit the bacterial enzyme DNA gyrase, which is essential for DNA replication. Hence, the present studies were initiated to determine whether the molecular species inhibiting the gyrase reaction was the monomeric or the micellar form. For this purpose, the rate of DNA replication was measured in a toluenized Escherichia coli cell system in the presence of increasing concentrations of novenamines. The resulting concentration-response curves proved anomalous, suggesting the involvement of micelles or some other, noncovalently aggregated forms of the inhibitors. The results were analyzed in terms of a variety of kinetic schemes and were found to be most consistent with the model where novenamines inhibit replicative DNA synthesis predominantly as cooperative dimers and, to a lesser extent, as monomers, but not as highly aggregated micelles. Based on this analysis and the knowledge that novobiocin and all novenamine-containing analogs are powerful gyrase inhibitors, we conclude that the target of the cooperative, dimeric inhibition is the gyrase, whereas the monomers of the novenamines inhibit another enzyme species involved in the bacterial DNA replication process.
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PMID:Novenamines as inhibitors of two independent enzymes during DNA replication in a toluenized Escherichia coli cell system. 878 54

Coinfection with mycoplasmas has been shown to enhance cytopathic changes in T lymphocytes in culture brought about by human immunodeficiency virus type-1 (HIV-1), accelerate disease progression, and suppress reverse transcriptase (RT) activity simultaneously. We attempted to identify the components in culture supernatants of mycoplasmas which suppress RT activity. The marked inhibitory effect on RT by culture supernatants was dependent upon Mg2+. The culture supernatants exhibited the activities of DNase and RNase, which degraded the products and substrates in RT assay, respectively. Gel filtration studies revealed that two major protein peaks, peak 1 (MW 67-100 kDa) and peak 2 (MW 10-25 kDa), exhibited DNase and/or RNase activities, and that both peaks contained a significant degree of inhibitory activity on RT. These results indicate that suppression of RT activity by the culture supernatants of mycoplasmas is due to DNase and RNase activities in the culture supernatants. The results of the present investigation suggest that RT assay of certain biological materials that are contaminated with mycoplasmas must be conducted carefully.
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PMID:Suppression of HIV-1 reverse transcriptase activity by culture supernatants of mycoplasmas. 878 58

Current research indicates that the nucleocapsid protein (NCp7) of human immunodeficiency virus type 1 (HIV-1) interacts with a variety of RNA substrates during the progression of the viral life cycle. The RNA features specifically recognized by the protein, however, have yet to be identified. SELEX was used to generate a set of RNAs whose affinities for nucleocapsid were on the order of 2 x 10(-9) M. Comparative analysis revealed that each RNA contains a highly conserved fourteen nucleotide sequence-block. Computer modeling and structure probing experiments indicate that the RNA ligands use the consensus sequence to fold into hairpins with an identical asymmetric bulge. The presence of the nucleocapsid protein protects the asymmetric bulge from ribonuclease attack, suggesting that it is the key element in protein recognition. A search for similar structural motifs within the HIV genome reveals several potential interaction sites for the nucleocapsid protein.
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PMID:A specific RNA structural motif mediates high affinity binding by the HIV-1 nucleocapsid protein (NCp7). 891 17

The RNA stem-loop structure of the trans-activating region TAR sequence of human immunodeficiency virus-1 mRNA is the binding site for a number of host cell proteins. A virtually identical set of proteins from HeLa nuclear extracts was found to bind to the predicted RNA hairpin element of prion protein (PrP) mRNA, as demonstrated in UV cross-linking/RNase protection and Northwestern assays. We show that the cellular TAR loop-binding protein, p68, is among those proteins which associate with PrP RNA. Competition experiments with various TAR RNA mutants revealed that binding of partially purified p68 to PrP RNA stem-loop occurs sequence-specifically. The 100-kDa 2-5A synthetase which is involved in the cellular antiviral defense was able to bind to PrP mRNA stem-loop in Northwestern blots with cytosolic proteins from HeLa cells treated with interferon. However, the PrP RNA failed to activate this enzyme in vitro, in contrast to TAR RNA.
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PMID:Interaction of 68-kDa TAR RNA-binding protein and other cellular proteins with prion protein-RNA stem-loop. 922 82


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