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)

A primary transcript from the chloroplast rpl32 gene was labelled at its 5' end using a capping enzyme and [alpha-32P]GTP followed by hybridization to a cold RNA probe. A RNase protection assay gave a clear protected band and its initiation site of transcription could thus be estimated, which had not been possible by using DNA probes. The combination of in vitro capping and RNase protection is an excellent method for mapping transcription initiation sites on the chloroplast genome and shows a high improvement relative to the DNA-employing strategies.
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PMID:Combination of in vitro capping and ribonuclease protection improves the detection of transcription start sites in chloroplasts. 162 81

According to the allosteric three-site model of the elongation cycle the ribosome oscillates between two main-functional states, viz the pre-translocational state with occupied A and P sites (E site with low affinity) and the post-translocational state with occupied P and E sites (A site with low affinity). This proposition could be confirmed by a determination of the thermodynamic parameters. High activation-energy barriers were found between both states, namely about 90 kJ mol-1 at 15 mM Mg2+ for either transition (post----pre transition = A-site binding and pre----post transition = translocation). The various A-site states (binding of ternary complex, EF-Tu dependent GTP cleavage, peptide-bond formation) are not separated by significant activation-energy barriers. The rate-limiting step of the elongation cycle is A-site binding, and not translocation as assumed previously. The principal role of both elongation factors is the reduction of the respective activation-energy barrier, thus accelerating the rate of the elongation cycle by several orders of magnitude. Cleavage of a single phosphodiester bond after G2661 of 23S rRNA by the RNase alpha-sarcin abolishes the functions of both elongation factors on the ribosome. This observation implies that the alpha-sarcin stem-loop structure plays an important role in the ribosomal conformational changes involved in the allosteric transitions. Indeed we could demonstrate that suitable oligodeoxynucleotide probes complementary to the alpha-sarcin region induce a conformational change in the 50S subunits; this conformational change causes an irreversible dissociation of tightly coupled ribosomes upon sucrose-gradient centrifugation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The two main states of the elongating ribosome and the role of the alpha-sarcin stem-loop structure of 23S RNA. 163 65

The incorporation of radiolabeled GTP into RNA in host-free Chlamydia trachomatis serovar L2 organisms was investigated. The incorporation was partially inhibited by rifampin and dactinomycin and hydrolyzed by RNase. RNA made by host-free chlamydiae consisted mainly of species of fewer than 800 bases in size, although 16S and 23S species were noted by agarose-gel electrophoresis. The hybridization of radiolabeled host-free RNA to restriction fragments of the gene encoding the major outer membrane protein was analyzed; all regions of the gene were transcribed. The relative intensity of hybridization of host-free RNA made by chlamydiae isolated during the middle and late stages of the developmental cycle to the DNA of clones encoding gene products known to be made at these times in vivo indicated that the temporal patterns of host-free and in vivo transcription were similar. Radiolabeled RNA from 1- and 24-h host-free Chlamydia psittaci 6BC organisms hybridized to many of the same EcoRI and BamHI restriction fragments of C. psittaci genomic DNA, although some differences could be noted. When these RNAs were used to screen a partial C. psittaci genomic library in lambda gt11, plaques were identified that reacted mainly either with 1-h RNA or with 24-h RNA. Because RNA synthesized by host-free chlamydiae appears to be developmental cycle stage specific, transcripts made by host-free chlamydiae may be convenient probes that can be used to clone developmental stage-specific chlamydial genes.
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PMID:Developmental cycle-specific host-free RNA synthesis in Chlamydia spp. 169 76

The stoichiometry of the EF-Tu-GTP-aminoacyl-tRNA complex has been re-determined by a variety of methods, viz gel filtrations, fluorescence titrations, as well as hydrolysis and RNase protection experiments. The results of these experiments clearly demonstrate that one aminoacyl-tRNA interacts with only one EF-Tu-GTP molecule, in agreement with the established view and in contrast to the recently published results by Ehrenberg et al [6].
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PMID:How many EF-Tu molecules participate in aminoacyl-tRNA binding? 174 49

Approximately one-third of the total ATP-hydrolysis activity in isolated HeLa nuclei is sensitive to RNAase (ribonuclease). This activity is selectively extracted with pulse-labelled RNA. In the extracts it co-sediments with various particles with sedimentation coefficients from 10S to 50S, but especially with 24S and 40S particles. ATP hydrolysis by the isolated particles was inhibited extensively (greater than 80%) by RNAase A, heparin and 0.2 M-NaCl. The activity of RNAase-treated particles was recovered when poly(A) was added, but not when DNA was added. The isolated particles exhibited RNAase-sensitive hydrolysis activities for dATP, GTP, CTP and UTP as well as for ATP, and the UTPase activity in the extracts showed nearly the same sedimentation distribution as the ATPase activity. When samples of isolated particles were irradiated with u.v. light in the presence of [alpha-32P]ATP, a 39 kDa polypeptide with a broad distribution from 10S to 50S like that of the ATPase and a 55 kDa polypeptide with a sharp distribution at 24S were photolabelled. Taken together, the data suggest that ATP-hydrolysis activity found in nuclear ribonucleoprotein subfractions appears to be the result of one or two RNA-dependent NTPases that are normally associated with endogenous RNA in a wide variety of particles.
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PMID:Characterization of a ribonuclease-sensitive nucleoside triphosphatase activity from HeLa nuclei. 240 2

More than 90% of rapidly-labelled nuclear RNA was associated with a nuclear matrix prepared from mouse leukemia L5178Y cells. The binding was not affected with up to 4 M NaCl; however, these RNAs were released from the nuclear matrix by treatment with a low ionic strength buffer (5 mM Tris-HCl buffer, pH 7.5, containing 1 mM ATP, 1 mM dithiothreitol, 0.2 mM ethylenediaminetetraacetic acid (EDTA) and 0.4 mM calcium chloride), without destruction of the sphere of the nuclear matrix. Actin filaments in the nuclear matrix were depolymerized with this buffer accompanied with rapidly-labelled RNAs. When the depolymerization was inhibited by slight modifications of the low ionic strength buffer (replacement of ATP by the same concentration of GTP; replacement of calcium ion by the same concentration of magnesium ion; addition of 20 micrograms/ml of phalloidine, which is a specific inhibitor of actin depolymerization), the release of rapidly-labelled RNAs from the nuclear matrix was also inhibited. The complex containing rapidly-labelled RNAs and matrix proteins was solubilized by a sonication from the nuclear matrix, and subjected to cesium chloride equilibrium centrifugation. Rapidly-labelled RNAs were concentrated on the bottom of the gradient accompanied with a small number of proteins (68K, 60K, 43K and 40K). The 43K protein was identified as actin by immunoblotting. By RNase digestion before equilibrium centrifugation, actin in the bottom fractions disappeared. These results suggest that rapidly-labelled RNAs anchor on the actin filaments in the nuclear matrix.
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PMID:Association of rapidly-labelled RNAs with actin in nuclear matrix from mouse L5178Y cells. 241 67

It has recently been shown that the non-formylated initiator Met-tRNAfMet from E. coli can form a stable ternary complex with the elongation factor EF-Tu and GTP. Using the protection of EF-Tu:GTP against spontaneous hydrolysis of the aminoacylester bond of Met-tRNAfMet, we confirm these results, and show that the protection is specific for the non-formylated form of the initiator tRNA. The ternary complex Met-tRNAfMet:EF-Tu:GTP can be isolated by column chromatography in a way similar to that demonstrated previously with EF-Tu complexed to the elongator Met-tRNAmMet. 32P-labeled Met-tRNAfMet within the ternary complex was analyzed by the footprinting technique. The pattern of initiator tRNA protection by EF-Tu against ribonuclease digestion is not significantly different from the one found previously for elongator tRNAs. These results lead us to suggest that the initiator tRNAfMet, under growth conditions which do not permit formylation, may to some extent function as an elongator tRNA.
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PMID:Interaction between initiator Met-tRNAfMet and elongation factor EF-Tu from E. coli. 242 55

We report studies in vitro of the interaction between non-formylated initiator Met-tRNA(fMet) and 70S ribosomes. The binding of Met-tRNA(fMet) to ribosomes carrying fMet-tRNA(fMet) in the P-site is strongly stimulated by elongation factor EF-Tu:GTP in the presence of (AUG)3. The enzymatically bound Met-tRNA(fMet) does not react with puromycin. The bound Met-tRNA(fMet) can accept formylmethionine from P-site-bound fMet-tRNA(fMet). These results demonstrate a functionally active binding at the ribosomal A-site. Partial ribonuclease digestion (footprinting) was used to study the sites in Met-tRNA(fMet) which are involved in the interaction with the ribosomal A-site. The results show that a large part of the tRNA molecule is protected by the ribosome against ribonuclease digestion. In addition to the protection found in the amino acid region and the anticodon arm, protection is seen in the D-loop and in the extra arm. No region within the bound tRNA is found to be more accessible for RNases than in the free Met-tRNA(fMet). The reported enhancement of ribonuclease cuts in the D- and T-arms of A-site-bound Phe-tRNAPhe is thus not found in A-site bound Met-tRNA(fMet).
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PMID:Interaction between non-formylated initiator Met-tRNA(fMet) and the ribosomal A-site from Escherichia coli. 244 56

Isolated rat liver nuclei were incubated under conditions when RNA polymerase I or RNA polymerase II was preferentially active. It was shown that [gamma-32P] ATP and [gamma-32P] GTP were incorporated into phenol extractable, TCA-precipitable material. RNase, actinomycin D, heparin and, in the case of RNA-polymerase II, alpha-amanitine inhibited precursor incorporation. These data are interpreted as evidence in favour of the initiation of RNA synthesis in isolated rat liver nuclei.
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PMID:[Initiation of RNA synthesis in isolated rat liver nuclei]. 258 May 66

Total RNA from Ehrlich ascites mitochondria pretreated with RNase-free DNase was capped in vitro with [alpha-32P]GTP and guanylyl transferase. The cappable RNAs representing the primary transcripts show a heterogeneous size distribution with four major species of 46, 63, 94, and 152 nucleotides and four minor species of 19, 24, 104, and 790 nucleotides in size. Hybridization with the D-loop DNA probes shows that the 19-nucleotide-long capped RNA is coded by the H-strand of mitochondrial DNA while the rest are coded by the L-strand. S1 nuclease mapping and primer extension analyses suggest the occurrence of a transcription initiation of H-strand at about 19 nucleotides upstream from the start of the tRNA(Phe) gene. All of the L-strand cappable RNAs have a common 5' end mapping to nucleotide 16,183 +/- 5 of the genome. The 3' ends of four major cappable RNA species line up to the conserved sequence boxes, putative start sites of DH-DNA; and in fact about 2% of these cappable species are found to exist as DNA-linked RNA under steady-state conditions. The 3' end of the 790-nucleotide cappable RNA lies close to the start of the tRNA(Pro) gene, suggesting that it may be the true precursor of L-strand transcript endonucleolytically processed at the 3' end. The level of L-strand-coded cappable RNAs varies markedly under different growth conditions. Treatment with cycloheximide results in a reduction while chloramphenicol caused over 3-fold induction, suggesting that these "primer" RNAs may have an additional regulatory function.
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PMID:Characterization of primary transcripts and identification of transcription initiation sites on the heavy and light strands of mouse mitochondrial DNA. 271 42


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