Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
 ...
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

One of the inherent problems in the use of antisense oligodeoxynucleotides to ablate gene expression in cell cultures is that the stringency of hybridization in vivo is not subject to control and may be sub-optimal. Consequently, phosphodiester or phosphorothioate antisense effectors and non-targeted cellular RNA may form partial hybrids which are substrates for RNase H. Such processes could promote the sequence dependent inappropriate effects recently reported in the literature. We have attempted to resolve this problem by using chimeric methylphosphonodiester/phosphodiester oligodeoxynucleotides. In contrast to the extensive RNA degradation observed with all-phosphodiester oligodeoxynucleotides, highly modified chimeric antisense effectors displayed negligible, or undetectable, cleavage at non-target sites without significantly impaired activity at the target site. We also note that all of the all-phosphodiester oligodeoxynucleotides tested demonstrated inappropriate effects, and that such undesirable activity could vary widely between different sequences.
 ...
PMID:Increased specificity for antisense oligodeoxynucleotide targeting of RNA cleavage by RNase H using chimeric methylphosphonodiester/phosphodiester structures. 137 64

Protected 2'-deoxy-2'-fluorouridine and 2'-deoxy-2'-fluorocytidine suitable for incorporation into oligonucleotides via the phosphoramidite approach have been prepared. Five modified and two unmodified oligonucleotides have been synthesized to investigate the regiospecific cleavage of a 5S RNA from Escherichia coli by RNase H. In order to show whether the modified oligonucleotides are able to hybridize with the RNA the physico-chemical properties (melting curves, CD spectra) of analogous DNA/oligodeoxyribonucleotide duplexes have been examined. The modified oligonucleotides are shown to form stable duplexes with a DNA-matrix which exist in an A-like form. Two of the modified probes containing four 2'-deoxy-2'-fluorocytidines or two 2'-deoxy-2'-fluorouridines direct the splitting by RNase H of only one phosphodiester bond of the RNA.
 ...
PMID:The use of oligonucleotide probes containing 2'-deoxy-2'-fluoronucleosides for regiospecific cleavage of RNA by RNase H from Escherichia coli. 137 35

A novel small nuclear ribonucleoprotein (snRNP) complex containing both U11 and U12 RNAs has been identified in HeLa cell extracts. This U11/U12 snRNP complex can be visualized on glycerol gradients, on native polyacrylamide gels, and by selection with antisense 2'-O-methyl oligoribonucleotides. RNase H-mediated degradation of the U12 snRNA confirmed a direct interaction between the U11 and U12 snRNPs. This snRNP complex is the first to be identified involving low-abundance snRNPs. Selection of the U11/U12 snRNP complex is sensitive to high salt, suggestive of a protein-mediated interaction. Secondary structure analyses revealed several regions of the U11 snRNP accessible for interaction with other RNAs or proteins but no detectable difference between the accessibility of these regions in the U11 monoparticle compared with the U11/U12 snRNP complex. There are also several accessible single-stranded regions in the U12 snRNP, and oligonucleotide-directed RNase H digestion identified nucleotides 28 to 36 of U12 as containing sequences required for the U11/U12 interaction. Both the U12 snRNP and the U11/U12 snRNP complex can be disrupted without altering the cleavage/polyadenylation activity of a nuclear extract.
 ...
PMID:The low-abundance U11 and U12 small nuclear ribonucleoproteins (snRNPs) interact to form a two-snRNP complex. 137 90

The spatial arrangement of subunits p51 and p66 of the HIV-1 reverse transcriptase and the position of the RNase H containing domain, p15, have been determined by means of neutron small-angle scattering. The reverse transcriptase (p66/p51) is a flat molecule, which can be approximated by an ellipsoid with the half axes of 5.2 nm, 4.8 nm and 1.4 nm. The two subunits p51 and p66 having a centre-to-centre distance of 3.3 +/- 0.3 nm are attached at their flat sides, slightly shifted sideways. The p15 domain is located at the long axis of the ellipsoidal reverse transcriptase having a distance of 5.0 +/- 0.5 nm to the centre of the p51d domain, which is part of the p66 subunit, and a distance of 5.3 +/- 1.2 nm to the centre of the neighbouring p51s subunit.
 ...
PMID:Domain structure of the human immunodeficiency virus reverse transcriptase. 137 48

A method for the rapid preparation of a defined substrate to monitor RNase H activity has been developed. Using this substrate, we have investigated the RNase H activities of the different forms of recombinant HIV-1 and HIV-2 reverse transcriptase (RT) in detail. As we report here, RNase H activity is associated only with the dimeric forms (p51/p66 or p66/p66) of the enzymes.
 ...
PMID:RNase H activity of HIV reverse transcriptases is confined exclusively to the dimeric forms. 137 72

32P-Labeled 70S ribosomes and polysomes were isolated from cultures of Escherichia coli and treated with the cross-linking reagent bis(2-chloroethyl)methylamine. Intermolecular 16S-23S RNA cross-linked complexes were separated from other products of the cross-linking reactions by a two-step sucrose density gradient centrifugation procedure and subjected to oligodeoxynucleotide-directed partial nuclease digestions with RNase H. Cross-linked RNA fragments released by such directed digests were resolved by two-dimensional gel electrophoresis and analyzed using classical oligonucleotide fingerprinting techniques. Two distinct intermolecular cross-links between the 16S and 23S RNA could be localized in this manner, involving positions 1408-1411 and 1518-1520 in the 16S RNA sequence and positions 1912-1920 in the 23S RNA sequence. These data provide the first direct topographical links between the RNA of the 30S and 50S subunits in the functional ribosome and, together with previous topographical data concerning the three-dimensional folding of the rRNA, demonstrate that there is a tight cluster at the ribosomal interface both of sites implicated in ribosomal function and of posttranscriptionally modified nucleotides in the rRNA.
 ...
PMID:Identification of intermolecular RNA cross-links at the subunit interface of the Escherichia coli ribosome. 137 17

A hybrid enzyme which site-specifically hydrolyzes RNA was created by covalently linking an oligodeoxyribonucleotide to Escherichia coli ribonuclease HI, an enzyme which specifically cleaves RNA moiety of DNA/RNA hybrids. A cysteine residue was substituted for Glu135 by site-directed mutagenesis in the mutant enzyme, in which all 3 free cysteine residues were replaced by alanine (Kanaya, S., Kimura, S., Katsuda, C., and Ikehara, M. (1990) Biochem. J. 271, 59-66), and coupled with a maleimide group, which is attached to the 5' terminus of the nonadeoxyribonucleotide (5'-GTCATCTCC-3') with a flexible tether. The resulting hybrid enzyme, d9-C135/RNase H, cleaved the phosphodiester bond between the fifth and sixth residues of the complementary nonaribonucleotide, without addition of the oligodeoxyribonucleotide. The nonaribonucleotide is cleaved by the wild-type or unmodified mutant enzyme only when the complementary oligodeoxyribonucleotide is present. When the kinetic parameters of the hybrid enzyme for the hydrolysis of the nonaribonucleotide were compared with those of the unmodified mutant enzyme for the hydrolysis of the nonanucleotide duplex, the hybrid enzyme exhibited a 7- and 4-fold decreases in the Km and kcat values, respectively, indicating that it performs multiple turnovers and has a sufficiently high hydrolytic activity. Hybrid ribonucleases H with various oligodeoxyribonucleotides in size and sequence, therefore, might be used as excellent tools for structural and functional studies of RNA.
 ...
PMID:A hybrid ribonuclease H. A novel RNA cleaving enzyme with sequence-specific recognition. 137 29

AIDS, caused by human immunodeficiency virus (HIV), is one of the world's most serious health problems, with current protocols being inadequate for either prevention or successful long-term treatment. In retroviruses such as HIV, the enzyme reverse transcriptase copies the single-stranded RNA genome into double-stranded DNA that is then integrated into the chromosomes of infected cells. Reverse transcriptase is the target of the most widely used treatments for AIDS, 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI), but resistant strains of HIV-1 arise in patients after a relatively short time. There are several nonnucleoside inhibitors of HIV-1 reverse transcriptase, but resistance to such agents also develops rapidly. We report here the structure at 7 A resolution of a ternary complex of the HIV-1 reverse transcriptase heterodimer, a monoclonal antibody Fab fragment, and a duplex DNA template-primer. The double-stranded DNA binds in a groove on the surface of the enzyme. The electron density near one end of the DNA matches well with the known structure of the HIV-1 reverse transcriptase RNase H domain. At the opposite end of the DNA, a mercurated derivative of UTP has been localized by difference Fourier methods, allowing tentative identification of the polymerase nucleoside triphosphate binding site. We also determined the structure of the reverse transcriptase/Fab complex in the absence of template-primer to compare the bound and free forms of the enzyme. The presence of DNA correlates with movement of protein electron density in the vicinity of the putative template-primer binding groove. These results have important implications for developing improved inhibitors of reverse transcriptase for the treatment of AIDS.
 ...
PMID:Structure of HIV-1 reverse transcriptase/DNA complex at 7 A resolution showing active site locations. 137 66

Human-immunodeficiency-virus-type-1 reverse transcriptase exists in virions as a heterodimer of a M(r) 66,000 subunit and its C-terminally truncated form of M(r) 51,000, but, when expressed as a recombinant M(r) 66,000 protein, a mixture of heterodimers and homodimers results which co-purify by most conventional techniques. We describe a method of hydrophobic chromatography which gives baseline separation of these two forms of the protein. This method has been applied to purify heterodimers formed by recombination of separately expressed and purified M(r) 66,000 and 51,000 subunits, resulting in significantly more homogeneous heterodimer preparations. The recombined heterodimer showed similar kinetic properties and RNase H activity to the standard heterodimer and a specific activity significantly higher than the original homodimer of the M(r) 66,000 protein. Heterodimers having greater asymmetry have also been prepared by recombining Mr 66,000 subunits containing single-point or deletion mutations, with wild-type M(r) 51,000 subunits, and the resulting heterodimers analysed.
 ...
PMID:Formation of heterodimers of human-immunodeficiency-virus-type-1 reverse transcriptase by recombination of separately purified subunits. 137 16


<< Previous 1 2 3 4 5 6 7 8 9 10