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:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An in vitro transcription system in which vesicular stomatitis virus (VSV) mRNA species have been synthesized is described. In addition to purified VSV virions, which contain an RNA-dependent RNA polymerase, this system contained a cytoplasmic cell extract that enhanced correct transcription. Gel electrophoretic analysis of the methylated polyadenylic acid [poly(A)]-containing VSV mRNA produced in this system in the presenct of S-adenosylmethionine showed the discrete VSV mRNA species. However, when unmethylated mRNA was synthesized in the presence of S-adenosylhomocysteine, the poly(A)-containing transcripts were large and heterogeneous in molecular weight and did not contain discrete VSV mRNA species. Two-dimensional fingerprint analysis of the methylated and unmethylated products suggested that identical nucleotide sequences were present in the RNAs. Further analysis showed the presence of very large heterogeneous poly(A), 200 to 2,000 nucleotides in lenght, in the unmethylated transcript. Proof that this large poly(A) was covalently linked to the correct VSV mRNA transcripts was obtained by removal of the poly(A) by hybirdization with oligodeoxythymidylic acid and digestion with RNase H. This digestion produced unmethylated VSV mRNA transcripts with the same discrete sizes as the deadenylated RNAs produced from VSV mRNA initially isolated from VSV-infected cells. The results suggest that there is a relationship between methylation at the 5'-end and polyadenylation at the 3'-end of VSV mRNA's. Furthermore, addition of the very large poly(A) does not affect the normal process of sequential transcription of the VSV genome, suggesting that this poly(A) addition is occurring independently of further transcription.
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PMID:Giant heterogeneous polyadenylic acid on vesicular stomatitis virus mRNA synthesized in vitro in the presence of S-adenosylhomocysteine. 18 93

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.
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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

The interaction of several forms (p51, p66, and p66/p51) of recombinant human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) with a synthetic derivative of its cognate replication primer, tRNA(Lys-3), has been determined by gel-mobility shift analysis. While p66/p51 RT is proficient in tRNA binding, preparations of p66 and p51 display only weak binding at elevated protein:tRNA ratios, despite the former containing both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activity. Gel permeation analysis of purified p66 RT indicate this to be predominantly monomeric, suggesting that dimerization may be a prerequisite for efficient tRNA binding. Prolonged incubation of a mixture of the 66- and 51-kDa polypeptides results in heterodimer reconstitution, restoration of tRNA binding, and recovery of appreciable levels of RNA-dependent DNA polymerase activity. Under the same conditions, both the tRNA binding and RNA-dependent DNA polymerase activities of the 66- and 51-kDa polypeptides are unaffected, suggesting that they remain in the monomeric conformation.
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PMID:Interaction of tRNA(Lys-3) with multiple forms of human immunodeficiency virus reverse transcriptase. 137 42

Native reverse transcriptase from simian immunodeficiency virus was purified from virus with good recovery to near homogeneity. The optimum reaction conditions of the enzyme were determined with respect to divalent cations, pH and ionic strength. The enzyme was shown to possess both RNA-dependent and DNA-dependent DNA synthesis activity. In addition, we could demonstrate an associated RNase H activity. Employing novel assay conditions, activated DNA as a heteropolymeric substrate was used more efficiently than the homopolymeric substrate poly(rA).oligo(dT) which in turn was used twofold more effectively as the template primer than poly(dC).oligo(dG). Other homopolymeric substrates, including poly(rC).oligo(dG), were also tested but were found to be poorly used by the reverse transcriptase. The Miachaelis-Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Simultaneously, using dideoxyadenosine triphosphate as nucleotide analogue, we could show that this compound acts as a competitive inhibitor with respect to dATP, whereas it acts as a non-competitive inhibitor with respect to the other nucleotides. Gel electrophoretic analysis showed the enzyme to consist of two polypeptides with apparent molecular masses of 64 and 48 kDa. Using activity gel electrophoresis, we were able to demonstrate that both subunits exhibit DNA synthesis activity.
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PMID:Simian immunodeficiency virus reverse transcriptase. Purification and partial characterization. 169 57

Poly(rA).oligo(dT)n binding to human immunodeficiency virus type-1 reverse transcriptase heterodimer (p66-p51) was primer length-dependent. The estimated Kd for (n = 10-14) was 20-30 nM and for (n = 16-20) was 0.11-0.14 nM. Gel electrophoretic analysis of the patterns of primer extension was consistent with an abrupt change in the Kd between a primer length of 14 and 16 nucleotides. Further, the rate constant for dissociation of the reverse transcriptase-template-primer complex was determined from steady state kinetics and enzyme-template-primer trapping experiments to be independent of primer length. Thus, the abrupt change in Kd was most likely due to a change in the rate constant for formation of the reverse transcriptase-template-primer complex. A similar shift in the Kd for template-primer binding was observed with poly(dA).oligo(dT)n. Reverse transcriptase homodimer (p66) catalyzed the incorporation of dTMP into poly(rA).oligo(dT)n with the same primer length dependence observed for the heterodimer. In contrast, binding of the p51 homodimer to poly(rA).oligo(dT)n was independent of primer length. Thus, the RNase H domain may contribute to reverse transcriptase heterodimer or p66 homodimer binding to template-primers in which the primer length is greater than 14 nucleotides.
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PMID:Human immunodeficiency virus reverse transcriptase. Effect of primer length on template-primer binding. 171 16

The inhibitor captan (N-trichloromethylthio-4-cyclohexen-1,2-dicarboximide) was used to explore the ribonuclease H (RNase H) active site of avian myeloblastosis virus (AMV) reverse transcriptase. Gel permeation chromatography of purified enzyme showed that [14C]captan bound to the alpha subunit in a ratio of 10:1 and to a 32,000 d polypeptide in a ratio of 4:1. Neither the alpha beta nor the beta subunit bound [14C]captan. The binding of 5 of the captan molecules was prevented by preincubating enzyme with polynucleotide. Deoxyguanosine triphosphate (dGTP) protected the enzyme against the binding of 4 captan molecules. Each holoenzyme bound 2 molecules of [3H]dGTP in the absence of, and 1 molecule of [3H]dGTP in the presence of 1 mM captan. Ribonuclease H activity was inhibited when AMV reverse transcriptase was preincubated with 1 mM captan before the degradative reaction was initiated. Preincubation of enzyme with polynucleotide before exposure to captan could partially protect the RNase H activity (61 +/- 2% activity remained). Deoxyguanosine triphosphate also partially protected the RNase H activity from inhibition by captan (75 +/- 9% activity remained). Inhibition of the RNase H activity was completely prevented by preincubating enzyme simultaneously with polynucleotide and dGTP. When separated by glycerol gradients the alpha subunit and alpha beta dimer both exhibited RNase H activity, but only the RNase H activity of the alpha subunit was inhibited by captan. Activity and binding studies revealed that the RNase H and polymerase activities of the alpha subunit are not susceptible to the interaction of captan when this subunit is in the alpha beta dimer form.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Captan binding to avian myeloblastosis virus reverse transcriptase and its effect on RNase H activity. 216 33

Biotinylation of phosphodiester oligodeoxynucleotides (PO-ODN) allows for conjugation to avidin-based transcellular delivery systems. In addition, biotinylation of PO-ODN at the 3'-terminus provides complete protection against serum 3'-exonuclease degradation. The present study was undertaken to determine if antisense 3'-biotinylated PO-ODN-avidin constructs are able to recognize and inactivate the target mRNA through RNase H-mediated degradation. A 21-mer antisense PO-ODN complementary to the tat gene encompassing nucleotides 5402-5422 of the HIV-1 genome was synthesized with biotin conjugated to the 3'-terminus (bio-tat). Gel mobility assays using [5'-32P]-labeled bio-tat ODN and avidin showed that the bio-tat ODN was fully monobiotinylated. Aliquots of [32P]-labeled sense or antisense tat RNA (337 and 351 nucleotides, respectively) were prepared from transcription plasmids and were preincubated with an excess of bio-tat ODN with or without avidin constructs and digested with RNase H. Products were resolved with sequencing gel and analyzed by autoradiography. Complete conversion to predicted RNA fragments resulting from RNase H digestion of the RNA-ODN duplex (53 and 263 nucleotides) was observed when [32P]-tat sense RNA was incubated with antisense bio-tat ODN or conjugated to avidin or an avidin-cationized human serum albumin (cHSA) complex. Conversely, no degradation of [32P]-tat-antisense RNA was observed after incubation with antisense bio-tat ODN and RNase H. In addition, the avidin-cHSA complex significantly increased (84-fold) the uptake of [32P]-internally labeled bio-tat ODN and its stability against cellular nuclease degradation in peripheral blood lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Complete inactivation of target mRNA by biotinylated antisense oligodeoxynucleotide-avidin conjugates. 784 69

Folate receptors (FR) are inversely regulated by the extracellular folate concentration at the translational level in cervical carcinoma cells. Accordingly, the potential for interaction of cis-elements in FR-alpha mRNA and trans-factors in these cells was determined. Gel-shift assays identified two signals that were specifically derived from the interaction of cytosolic proteins with the 5'-untranslated region of FR-alpha mRNA. RNase T1 mapping revealed that the RNA sequences interacting with these proteins were located between nucleotides -133 to -116 (18-bases) and -158 to -116 (43-bases), upstream of the translation start site. However, selective RNase H cleavage indicated that the 18-base RNA sequence was the cis-element. The RNA-protein interaction was competed by poly(C), but not by poly(U), homopolymers. UV cross-linking and Northwestern blot analysis confirmed that the trans-factors were 46-kDa proteins. An 18-base antisense oligodeoxynucleotide complementary to the cis-element specifically quenched the RNA-protein interaction and also completely inhibited translation of FR-alpha mRNA without changing its stability. Thus, the interaction of the 18-base cis-element and the 46-kDa trans-factors likely have an important role in translational regulation of FR. In addition, because the 46-kDa proteins were widely distributed in cells expressing little to no FR-alpha, these species probably have additional functions that are unrelated to translation of FR.
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PMID:Evidence that a specific interaction between an 18-base cis-element in the 5'-untranslated region of human folate receptor-alpha mRNA and a 46-kDa cytosolic trans-factor is critical for translation. 881 Mar 26

Antisense oligonucleotides (ODNs) overlapping the stem-loop structure of the trans-activating responsive (TAR) element at the 5' end of HIV-1 and HIV-2 viral RNAs were tested for their inhibitory effect on cDNA synthesis by HIV-1 and HIV-2 reverse transcriptases (RT). Inhibition of reverse transcription is sequence-specific and enhanced by the presence of the RT-associated RNase H activity. The degree of inhibition obtained with the anti-TAR antisense is significantly higher than with other HIV-1 targeted antisense ODNs used before [1]. Gel retardation showed a stable specific complex between the 16- and 25-mer anti-TAR HIV-1 selected ODNs and the target region. No complex was observed with a non-inhibitor 22-mer anti-TAR ODN and with the corresponding control sequences. Targeting of the first stem-loop in the 5' region of HIV-2 RNA by anti-TAR ODNs inhibited very strongly reverse transcription by HIV-2 RT. The structure of the antisense and the target sequence affect annealing efficiency and hence the degree of inhibition of reverse transcription.
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PMID:Specific inhibition of in vitro reverse transcription using antisense oligonucleotides targeted to the TAR regions of HIV-1 and HIV-2. 913 May 87

Frontotemporal dementia accounts for a significant fraction of dementia cases. Frontotemporal dementia with parkinsonism linked to chromosome 17 is associated with either exonic or intronic mutations in the tau gene. This highlights the involvement of aberrant pre-mRNA splicing in the pathogenesis of neurodegenerative disorders. Little is known about the molecular mechanisms of the splicing defects underlying these diseases. To establish a model system for studying the role of pre-mRNA splicing in neurodegenerative diseases, we have constructed a tau minigene that reproduces tau alternative splicing in both cultured cells and in vitro biochemical assays. We demonstrate that mutations in a nonconserved intronic region of the human tau gene lead to increased splicing between exon 10 and exon 11. Systematic biochemical analyses indicate the importance of U1 snRNP and, to a lesser extent, U6 snRNP in differentially recognizing wild-type versus intron mutant tau pre-mRNAs. Gel mobility shift assays with purified U1 snRNP and oligonucleotide-directed RNase H cleavage experiments support the idea that the intronic mutations destabilize a stem-loop structure that sequesters the 5' splice site downstream of exon 10 in tau pre-mRNA, leading to increases in U1 snRNP binding and in splicing between exon 10 and exon 11. Thus, mutations in nonconserved intronic regions that increase rather than decrease alternative splicing can be an important pathogenic mechanism for the development of human diseases.
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PMID:Aberrant splicing of tau pre-mRNA caused by intronic mutations associated with the inherited dementia frontotemporal dementia with parkinsonism linked to chromosome 17. 1080 46


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