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)

Activities of the hepadnavirus polymerases are known to include those of DNA polymerase, reverse transcriptase and RNase H. To date, it has been difficult or impossible to clone and express the product as an active enzyme. In this study, full length capped RNA encoding Duck Hepatitis B Virus (DHBV) polymerase was produced by in vitro transcription from a T7 promoter. The RNA was translated in a rabbit reticulocyte lysate system and produced an 35S-Methionine labelled 79 Kd band on SDS-polyacrylamide gel electrophoresis. The translation product showed DNA polymerase and reverse transcriptase activities on exogenous templates (respectively) of DNA or RNA with random DNA hexamer primers. The same RNA transcripts were also microinjected into Xenopus oocytes, but appeared to be toxic and gave no detectable translation product. Production of hepadnavirus polymerase by in vitro transcription/translation may provide a useful tool for structure/function and pharmacological studies on this important group of polymerases.
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PMID:Duck hepatitis B virus polymerase produced by in vitro transcription and translation possesses DNA polymerase and reverse transcriptase activities. 128 90

The reverse transcriptase of human immunodeficiency virus type 1 is a heterodimeric protein consisting of two polypeptides with masses of 66 and 51 kDa and has, as a second enzymatic activity, RNase H activity. The 66-kDa polypeptide can be cleaved by the virus-encoded protease to yield polypeptides of 51 and 15 kDa. The latter has been characterized as possessing RNase H activity [Hansen, J., Schultze, T., Mellert, W. & Moelling, K. (1988) EMBO J. 7, 239-243]. We have purified simultaneously the heterodimeric reverse transcriptase/RNase H containing the 66/51-kDa polypeptides and the 15-kDa RNase H from Escherichia coli containing the expression vector pJS 3.7 by a procedure including chromatography on DEAE-cellulose, phosphocellulose, and heparin-Sepharose. Two RNase H and reverse transcriptase peaks were separated on phosphocellulose, one coinciding with the heterodimeric protein and the other with the 15-kDa protein. On the basis of the following findings it appears that the 15-kDa polypeptide has both RNase H and reverse transcriptase activities: (i) it copurified with both activities; (ii) it functioned as a reverse transcriptase in an in situ assay after SDS/polyacrylamide gel electrophoresis; (iii) polyclonal antibodies raised against the 66-kDa polypeptide reacted in immunoblots exclusively with a 15-kDa polypeptide, reacted in immunoblots exclusively with a 15-kDa polypeptide, while no immunoreactive bands in the range of 51-66 kDa were seen in the 15-kDa polypeptide preparation; (iv) the p15 and the p66/51 reverse transcriptase could be quantitatively pelleted in an enzymatically active form only when antibodies specific for the p66 carboxyl terminus were used; and (v) the p15 protein had bona fide properties of a reverse transcriptase and could enzymatically synthesize a high molecular weight, alkali-resistant product. The two reverse transcriptases appear to have different behaviors on various template/primer systems tested. Conceivably different forms of human immunodeficiency virus type 1 reverse transcriptases might be used in individual steps of (+)- and (-)-strand replication.
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PMID:The p15 carboxyl-terminal proteolysis product of the human immunodeficiency virus type 1 reverse transcriptase p66 has DNA polymerase activity. 171 Dec 22

The reverse transcriptase/RNase H of HIV-1 is composed of a p66/p51 heterodimer when analyzed from virus particles. A recombinant reverse transcriptase (RT)/RNase H which after purification consisted mainly of p66 was analyzed as substrate of the purified recombinant HIV-1 protease p9 in vitro. The p66 protein if treated with the protease is processed to a stable p66/p51 heterodimer. A p15 protein is a prominent cleavage product which was identified as the carboxyterminal portion of p66 by means of a monoclonal antibody. It exhibits RNase H activity when tested by activated gel analysis. Presence of SDS during the incubation allowed complete degradation of p66 depending on the conditions, which indicates that conformation of a substrate is relevant for cleavage by the HIV-1 protease. A synthetic heptapeptide AET-FYVD derived from the region between RT and RNase H is cleaved efficiently in vitro by the HIV-1 protease at the F'Y junction, and may mimick a natural cleavage site. P66/p51 heterodimers exhibit higher RT and RNase H activities than p66 when renatured from polyacrylamide gels.
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PMID:Cleavage of the HIV-1 p66 reverse transcriptase/RNase H by the p9 protease in vitro generates active p15 RNase H. 171 81

Destabilizing events required for subsequent cotranslational disassembly of tobacco mosaic virus (TMV) particles in vitro were studied. Brief treatment of U-32P-labelled TMV (strain vulgare or U2) with 1% SDS exposed only 2.5% of the RNA (160 5' nucleotides) in a susceptible subpopulation of virions. Limited uncoating occurred almost immediately and appeared to be synchronous because the amount of 5' oligonucleotide marker (omega) recovered remained constant throughout a 15 min period in SDS. Additional RNase T1-sensitive oligonucleotides were exposed only after 1 to 2 min in SDS. Coat protein (CP) subunits released from virions 'destabilized' by ultracentrifugation at between pH 7.2 and 9.2 were quantified using L-[35S]methionine-labelled particles of TMV strain U2. CP recovery and virus particle translation results were consistent with increasing numbers of virions uncoating for approximately 200 nucleotides. In the presence of sparsomycin (SPN), the TMV strain vulgare 5' leader and the first AUG codon can bind two 80S ribosomes. Electron microscopy of pH 7.5-treated TMV particles incubated in SPN-treated wheatgerm extract or rabbit reticulocyte lysate, showed that approximately 10% of virions complexed with one ribosome and approximately 10% with two bound ribosomes, confirming that omega at least had been uncoated. Nucleocapsids in these complexes were shorter than untreated TMV by 9 to 10 nm (i.e. equivalent to 192 to 217 nucleotides exposed). The template activities of virions pretreated at pH 7.2 to 9.2 were destroyed by RNase H when short cDNAs were hybridized to sequences at, or immediately 3' to, the first AUG codon. We propose that the complete 5' leader of TMV RNA interacts weakly with CP subunits and that this micro-instability is due to the absence of G residues and is essential for initiation of cotranslational virus disassembly.
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PMID:Complete uncoating of the 5' leader sequence of tobacco mosaic virus RNA occurs rapidly and is required to initiate cotranslational virus disassembly in vitro. 184 66

We have reinvestigated the molecular weight and subunit composition of calf thymus ribonuclease H1. Earlier studies suggested a variety of molecular weights for the enzyme in the range of 64K-84K and reported that the enzyme either was a single polypeptide of 74 kDa or consisted of from two to four subunits in the range of 21-34 kDa. Although we too find bands in this lower molecular weight range in our highly purified preparations following SDS-PAGE, our data suggest that the native structure of RNase H1 is a dimer of 68-kDa subunits. The evidence includes the following: (1) Western blot analysis of fractions taken at various stages of the purification indicates that the predominant antigenic form of the enzyme in crude extracts has a molecular weight of 68K but that during purification in the absence of sufficient protease inhibitors a variety of lower molecular weight forms appear concomitant with the disappearance of the 68-kDa band. (2) Activity gel analysis of the highly purified enzyme prepared in the presence of a battery of protease inhibitors reveals that the 68-kDa band (as well as several bands of lower molecular weight) possesses RNase H activity. (3) The 68-kDa band recognized by Western blotting with anti-RNase H immune sera is not detected by using preimmune sera. Furthermore, when immune sera are used, a trace of a 140-150-kDa antigenic form can sometimes be detected, consistent with the existence of a dimeric form of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:On the molecular weight and subunit composition of calf thymus ribonuclease H1. 215 45

The avian RNA tumour virus structural protein p12 was purified from avian myeloblastosis virus (AMV) by nucleic acid affinity chromatography to apparent homogeneity as judged from SDS--polyacrylamide gel electrophoresis. A filter binding assay was used for the identification of p12. High concentrations of p12 precipitated nucleic acids out of solution in the absence of MgCl2. Binding of p12 to single-stranded nucleic acids protected them from digestion with nucleases and resulted in a hyperchromic effect. These phenomena were reversible in the presence of salt. The affinity of p12 to nucleic acids was determined by competing for the binding of p12 to denatured radioactive DNA by various other nuclei acids. It was found that p12 bound preferentially to single-stranded nucleic acids and showed a higher affinity to poly(rI) than to poly(rC) and poly(rA). Purified RNA-dependent DNA polymerase activity from AMV was stimulated up to sixfold by p12, depending on the template. Solubilization of RNA in RNA--DNA hybrids by RNase H was inhibited in the presence of p12.
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PMID:Properties of the avian viral protein p12. 616 96

We have mapped specific RNA-protein contacts between human immunodeficiency virus (HIV) type I reverse transcriptase (RT) and its natural primer, human tRNA(3Lys), using a site-specific crosslinking strategy. Four different tRNA(3Lys) constructs with a single 32P-labeled 4-thiouridine (4-thioU) residue at positions -1, 16, 36 or 41 were synthesized. After incubation with RT followed by irradiation, crosslinks were localized to either the p66 or p51 subunit of RT by digestion with nuclease and SDS gel fractionation. 4-thioU at position -1 or 16 transferred label to the p66 subunit almost exclusively (> 90%), whereas position 36 labeled both p66 and p51 (3:1). Position 41 yielded no detectable crosslinks. The region of p66 contacted by position -1 of tRNA(3Lys) was localized to the 203 C-terminal amino acids of RT by CNBr cleavage, whereas a 127 amino acid-CNBr peptide (residues 230-357) from both p66 and p51 was labeled by position 36. Functionality of the 4-thioU-modified tRNA(3Lys)(-1) crosslinked to RT in the presence of an RNA but not a DNA template was demonstrated by the ability of the tRNA to be extended. These results localize the 5' half of the tRNA on the interface between the two RT subunits, closer to the RNase H domain than to the polymerase active site, in accord with previous suggestions. They argue further that a specific binding site for the 5' end of the primer tRNA(3Lys) may exist within the C-terminal portion of the p66 subunit, which could be important for the initiation of reverse transcription.
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PMID:Site-specific crosslinking of 4-thiouridine-modified human tRNA(3Lys) to reverse transcriptase from human immunodeficiency virus type I. 754 Jan 37

Reduced oxygen tension (hypoxia) induces a 3-fold increase in stability of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell line. To investigate the possibility that RNA-protein interactions are involved in mediating this increase in stability, RNA gel shift assays were performed using different fragments of labeled TH mRNA and the S-100 fraction of PC12 cytoplasmic protein extracts. We identified a sequence within the 3'-untranslated region of TH mRNA that binds cytoplasmic protein. RNase T1 mapping revealed that the protein was bound to a 28 nucleotide long sequence that is located between bases 1551-1579 of TH mRNA. Moreover, protein binding to this fragment was prevented with an antisense oligonucleotide directed against bases 1551-1579 and subsequent RNase H digestion. This fragment of the 3'-untranslated region of TH mRNA is rich in pyrimidine nucleotides, and the binding of cytoplasmic protein to this fragment was reduced by competition with other polypyrimidine sequences including poly(C) but not poly(U) polymers. The binding of the protein to TH mRNA was increased when cytoplasmic proteins were extracted from PC12 cells exposed to hypoxia (5% O2) for 24 h. Electrophoresis of the UV cross-linked RNA-protein complex on SDS-polyacrylamide gel electrophoresis revealed a complex of 74 kDa. The potential role of this protein-TH mRNA interaction in regulation of TH mRNA stability during hypoxia is discussed.
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PMID:Hypoxia stimulates binding of a cytoplasmic protein to a pyrimidine-rich sequence in the 3'-untranslated region of rat tyrosine hydroxylase mRNA. 790 89

The expression of genes encoding self-incompatibility ribonucleases (S-RNases) in Nicotiana alata were examined at both protein and RNA level for organ specificity. S-RNases recovered from stigmas and styles were indistinguishable by SDS-PAGE, chromatographic behaviour and RNase specific activity. The pistil S transcripts are heterogeneous in size, the stigma transcript being shorter and more heterogeneous than the transcripts in the style and ovary. RNase H analysis shows that this organ-specific difference is mainly in the length of the polyadenylate tail. By sequence analysis of cloned cDNAs we show that the transcript present in the stigma is derived from the same gene as the transcript in the style.
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PMID:S-locus products in Nicotiana alata pistils are subject to organ-specific post-transcriptional processing but not post-translational processing. 809 64

We are examining the spatial arrangement of ribosomal components in the vicinity of rRNA sequences of particular significance for ribosome structure and function through the use of radioactive, photolabile derivatives of oligoDNAs complementary to such sequences. The oligoDNA probes bind to their target sequences in intact ribosomal subunits, and, on photolysis, incorporate into neighboring ribosomal components. Labeled ribosomal proteins are subsequently identified by SDS-PAGE, RP-HPLC and immunoprecipitation. Labeled rRNA is identified at the nucleotide level by RNase H cleavage and primer extension. The results obtained provide a description of the neighborhood surrounding a particular rRNA sequence and impose important constraints on the evolving three-dimensional models of both the 30S and 50S subunits, since they typically oblige several ribosomal components to fall within defined distances of the targeted rRNA. Our results will be discussed in terms of these models, emphasizing their consistency or inconsistency.
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PMID:Photolabile oligoDNA probes of internal Escherichia coli ribosomal structure. 864 99


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