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)

Our earlier work on the recognition of Q beta plus strand RNA by replicase had shown by RNase degradation and by electron microscopic techniques that specific binding interactions occurred at two internal sites, the S-site and the M-site, but not at the 3'-end, i.e. the site of initiation of synthesis. Using essentially similar methods, we have found now for binding complexes of replicase with the minus strand a completely different pattern, namely considerable terminal binding, whereas binding to internal sites was without detectable specificity. In the case of plus strand complexes, simultaneous binding at the two internal sites and at a terminal site could be demonstrated by electron microscopy after initiation of RNA synthesis in the presence of host factor, GTP and ATP. A variant plus strand RNA containing a 490 nucleotide duplication near the 5'-end resulted in similar double-looped complexes, however with an elongated free arm, showing that the protein-bound terminal site was the 3'-end of the RNA. Interestingly, the same two-looped structures were also found for complexes consisting of plus strand RNA and host factor without replicase. This suggests that the role of the host factor on the plus strand template is to bring the 3'-end into the proximity of the S-site/M-site domain, where replicase can initiate on it. In contrast, the 3'-end of the minus strand appears to be directly available to the enzyme.
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PMID:Different mechanisms of recognition of bacteriophage Q beta plus and minus strand RNAs by Q beta replicase. 834 21

The cucumber mosaic virus (CMV) 3a movement protein (MP) was compared directly to the well-characterized tobacco mosaic virus (TMV) 30K MP by cloning the genes encoding these proteins into Escherichia coli, isolating the E. coli-expressed MPs, and characterizing them with regard to RNA- and NTP-binding activities. The two MPs were shown to bind single-stranded RNA and DNA cooperatively, but with no sequence specificity. However, discrete lengths of CMV RNA 3 could be protected against RNase digestion by the CMV 3a protein, indicating that the RNA was not uniformly covered by the MP after cooperative binding. The TMV 30K:RNA complex was more stable in NaCl than the CMV 3a:RNA complex; about 50% of the corresponding complexes were stable in 0.6 and 0.4 M NaCl, respectively. Both MPs could bind GTP strongly and UTP weakly, but not ATP or CTP. The CMV 3a protein expressed either in E. coli or in planta from RNA 3 of CMV was tagged at its C-terminus with six histidine residues, which facilitated its purification by affinity chromatography on a matrix containing Ni(2+)-nitrilotriacetate. The soluble, His-tagged 3a proteins, affinity-purified from E. coli and zucchini squash, both were able bind CMV RNA 3 in vitro.
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PMID:Comparison of the nucleic acid- and NTP-binding properties of the movement protein of cucumber mosaic cucumovirus and tobacco mosaic tobamovirus. 861 8

RNase protection assays were used in a comparative analysis of the quantities of mRNA for five "calcium-sensitive" (types I, III, V, VI, and VIII) adenylyl cyclases and one "calcium-insensitive" (type II) adenylyl cyclase in mouse cerebral cortex, cerebellum, and nucleus accumbens. The mRNA levels for type V adenylyl cyclase were dominant in the nucleus accumbens. Type V adenylyl cyclase mRNA was also found in the cerebral cortex and at low levels in the cerebellum. Type I adenylyl cyclase mRNA was the major form in the cerebellum with 15-50-fold higher levels compared with other adenylyl cyclase mRNAs. Type I adenylyl cyclase mRNA was also the most prominent adenylyl cyclase mRNA in the cerebral cortex, although the mRNA levels of other adenylyl cyclase forms were more comparable to those of the type I enzyme in this brain area. The mRNA levels for adenylyl cyclase types II, III, VI, and VIII were intermediate to low depending on the brain area. Cell membranes from the nucleus accumbens demonstrated adenylyl cyclase activity that was synergistically activated by concomitant addition of GTP and forskolin to assay mixtures, reflecting a characteristic of type V adenylyl cyclase protein. Calcium/calmodulin stimulated adenylyl cyclase activity in membranes from all three brain areas. However, synergistic activation of adenylyl cyclase activity by GTP and calcium/calmodulin was noted only with cortical membranes, and this characteristic may reflect the presence of type VIII adenylyl cyclase mRNA in the cortex. Although mRNA for type VIII adenylyl cyclase was almost equivalent in the cortex and cerebellum, the lack of a synergistic effect of GTP plus calcium/calmodulin on the cerebellar enzyme activity may be a result of the significant dominance of type I adenylyl cyclase mRNA (and protein) in the cerebellum. In general, the mRNA levels for the various adenylyl cyclases were predictive of the regulatory characteristics of adenylyl cyclase activity in membranes of the brain areas studied.
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PMID:Adenylyl cyclases: mRNA and characteristics of enzyme activity in three areas of brain. 866 89

In order to provide evidence for a potential role of heterotrimeric GTP-binding proteins in the transduction of developmental signals, we prepared cDNAs from Xenopus laevis embryos and looked for fragments amplified between primers located in conserved sequences of the different subtypes of beta subunit. Using the amplified fragment as a probe, we cloned a member of the beta subunit family. The deduced protein sequence of the amphibian cDNA is highly homologous to the beta 1 subtype and, accordingly, we have named the Xenopus gene XG beta 1. In situ hybridization and RNase protection assay revealed that XG beta 1 mRNA is confined to the animal hemisphere of the mature oocyte. This localization of XG beta 1 mRNA is established at stage V during oogenesis. Following fertilization, the maternal mRNAs cosegregate with animal cells during cleavage stages. At gastrulation, transcripts are expressed in the dorsal ectoderm layer that will give rise to the central nervous system. Thus, XG beta 1 mRNA belongs to the small family of localized maternal mRNAs; as a transducing protein, its restriction to a subset of embryonic cells could mediate the distinct responsiveness which contributes to the patterning of the embryo.
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PMID:The mRNA encoding a beta subunit of heterotrimeric GTP-binding proteins is localized to the animal pole of Xenopus laevis oocyte and embryos. 895 92

Labeling of 21-kDa material was observed when bovine brain soluble fraction was incubated with [adenylate-32P]NAD+ in the presence of GTP. The 21-kDa substrate, slightly smaller than C3 substrate in size, was labeled even without C3 exoenzyme. GTP could be replaced by nucleoside triphosphates other than ATP while ATP inhibited the GTP-induced labeling of 21-kDa substrate. After incubation of the soluble fraction with [adenylate-32P]NAD+ in the presence of GTP, [32P]ADP and [32P]ATP were detected in addition to [32P]AMP and [32P]ADP-ribose while only the last two nucleotides were observed without GTP. The 21-kDa substrate was labeled with [alpha-32P]ATP even in the absence of GTP, suggesting adenylylation rather than ADP-ribosylation. The labeled 21-kDa substrate, was extractable by phenol, disappeared with RNase treatment but not with tryptic digestion. Alkaline treatment of the phenol extract yielded an equal mixture of 3'-[32P]CMP and 2'-[32P]CMP. From these results we concluded that the 21-kDa labeling is a result of tRNA tailing with [alpha-32P]ATP generated from the [32P]AMP moiety of [adenylate-32P]NAD+. Results from reconstitution experiments using enzymes and tRNA purified from bovine brain soluble fraction, which are involved in this pathway, confirmed our conclusion.
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PMID:GTP-dependent modification of a 21-kDa substrate with NAD+ in bovine brain soluble fraction is not ADP-ribosylation of small G-protein but tailing of tRNA. 935 90

Experimental conditions for poly(G) synthesis from GTP on a poly(C) template with the aid of Escherichia coli DNA-dependent RNA polymerase were investigated. The reaction product was purified without the use of RNase. On the basis of spectral data, gel permeation chromatography, affinity adsorption and electron microscopic visualization, the poly(G) x poly(C) product was assumed to possess a high degree of structural regularity. Its in vitro and in vivo antiviral activities were compared with those of traditional poly(G) x poly(C) and poly(I) x poly(C). Template-dependent poly(G) x poly(C) was similar in its in vitro activity to poly(I) x poly(C) or even surpassed it, whereas the 'traditional' poly(G) x poly(C) was only slightly active in vitro. However, 'traditional' poly(G) x poly(C) and poly(I) x poly(C) had similar activity in vivo, whereas template-dependent poly(G) x poly(C) was much less active in vivo. The role of intramolecular structural regularity in the in vitro and in vivo antiviral activity of polyribonucleotide duplexes is discussed.
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PMID:Template-dependent biosynthesis of poly(G) x poly (C) and its antiviral activity in vitro and in vivo. 970 75

The hyphomycete Hirsutella thompsonii produces an extracellular insecticidal protein, Hirsutellin A. This basic protein, cytolytic against insect cells and capable of inhibiting protein translation, possesses biological features similar to the well-characterized ribosomal-inhibiting proteins (RIPs) alpha-sarcin, mitogellin, and restrictocin. Cloning and DNA sequencing analysis of the 3' and 5' RACE products of HtA cDNA identifies a consensus DNA sequence which encompasses the complete open reading frame of the HtA gene. This gene codes for a precursor of 164 aa which includes a 34-aa leader sequence. The leader sequence of HtA, like those found in RIPs, contains a signal and a pro sequence. The mature 130-aa HtA, having a calculated Mr = 14,159 and pI = 9.21, is considered a stable hydrophilic protein. HtA does not possess the characteristic RNase motif of fungal RIPs but does possess a series of consensus phosphorylation and myristoylation sites and a putative ATP/GTP binding site. The sequence of HtA is unique and does not produce the secondary or tertiary structures characteristic of other fungal RIPs. Copyright 1998 Academic Press.
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PMID:Cloning and sequencing of cDNA of the insecticidal toxin hirsutellin A 978 48

A ribonuclease protection assay was used to determine the equilibrium dissociation constants (Kd) for the binding of various RNAs by wheat germ EF-1alpha.GTP. Aminoacylated fully modified tRNAs and unmodified tRNA transcripts of four specificities (valyl, methionyl, alanyl, and phenylalanyl) from higher plants or Escherichia coli were bound with Kd values between 0.8 and 10 nM. A valylated 3'-fragment of turnip yellow mosaic virus RNA, which has a pseudoknotted amino acid acceptor stem, was bound with affinity similar to that of Val-tRNAVal. Uncharged tRNA and initiator Met-tRNAMet from wheat germ, RNAs that are normally excluded from the ribosomal A site in vivo, bound weakly. The discrimination against wheat germ initiator Met-tRNAMet was almost entirely due to the 2'-phosphoribosyl modification at nucleotide G64, since removal resulted in tight binding by EF-1alpha.GTP. A 44-nucleotide RNA representing a kinked acceptor/T arm obtained by in vitro selection to bacterial EF-Tu formed an Ala-RNA.EF-1alpha.GTP complex with a Kd of 29 nM, indicating that much of the binding affinity for aminoacylated tRNA is derived from interaction with the acceptor/T half of the molecule. The pattern of tRNA interaction observed for EF-1alpha (eEF1A) therefore closely resembles that of bacterial EF-Tu (EF1A).
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PMID:Quantitative assessment of EF-1alpha.GTP binding to aminoacyl-tRNAs, aminoacyl-viral RNA, and tRNA shows close correspondence to the RNA binding properties of EF-Tu. 987

In Scrobicularia plana testis, a nuclear acid phosphatase (ACPase) activity was detected in mid and late spermatids with the improved Gomori-chloride procedure. Lead deposits were first observed in mid spermatids at focal points over condensed chromatin strands, increasing in density as chromatin further condensated. In late spermiogenesis, lead deposits became concentrated between chromatin aggregates, and after total DNA compaction were transfered to the nuclear periphery and then shed into the cytoplasm. The specificity of the nuclear ACPase was tested against different pH values (3.9, 7.2, 7.8, 9.0), substrates (TPP, IDP, TMP, p-NCS, ATP, GTP, AMP, ADP, AMP-PNP) and inhibitors (NaF, levamisole, Zn, vanadate, theophylline). To further specify the nature of this nuclear ACPase, other enzymes were comparatively studied at their optimal pH values and at pH 5.0: nucleoside-diphosphatase, thiamin-pyrophosphatase, inorganic trimetaphosphatase, lysosomal arylsulfatases A and B, ATPase, GTPase, 5'-nucleotidase, adenylate kinase, and adenylate cyclase. Several other controls were introduced to exclude artefactual deposits induced by lead ions and tissue molecules. The results showed that the enzyme has an optimal pH at 5.0, a high specific affinity for beta-GP, and is inhibited by NaF, which suggests that it behaves as a type B-ACPase, and all controls demonstrated the specificity of the enzymic activity. Because lead deposits were specifically and temporally associated with spermatid chromatin condensation, when DNA and RNA synthesis, histones, phosphoproteins and RNA molecules strongly decrease, it is possible to suggest that the nuclear ACPase could be associated with DNA processing during chromatin compaction or involved in the hydrolysis of 2' and 3' nucleotides resulting from nuclear RNase action during RNA degradation.
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PMID:Chromatin condensation during Scrobicularia plana spermiogenesis: a controlled and comparative enzymatic ultracytochemical study. 1079 22

Hepatitis C virus translation is initiated on a approximately 330-nucleotide (nt)-long internal ribosomal entry site (IRES) at the 5' end of the genome. In this process, a 43S preinitiation complex (comprising a 40S ribosomal subunit, eukaryotic initiation factor 3 (eIF3), and a ternary [eIF2-GTP-initiator tRNA] complex) binds the IRES in a precise manner so that the initiation codon is placed at the ribosomal P site. This binding step involves specific interactions between the IRES and different components of the 43S complex. The 40S subunit and eIF3 can bind to the IRES independently; previous analyses revealed that eIF3 binds specifically to an apical half of IRES domain III. Nucleotides in the IRES that are involved in the interaction with the 40S subunit were identified by RNase footprinting and mapped to the basal half of domain III and in domain IV. Interaction sites were identified in locations that have been found to be essential for IRES function, including (i) the apical loop residues GGG(266-268) in subdomain IIId and (ii) the pseudoknot. Extensive protection from RNase cleavage also occurred downstream of the pseudoknot in domain IV, flanking both sides of the initiation codon and corresponding in length to that of the mRNA-binding cleft of the 40S subunit. These results indicate that the 40S subunit makes multiple interactions with the IRES and suggest that only nucleotides in domain IV are inserted into the mRNA-binding cleft of the 40S subunit.
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PMID:An enzymatic footprinting analysis of the interaction of 40S ribosomal subunits with the internal ribosomal entry site of hepatitis C virus. 1086 33


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