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)

We have recently proposed that aminoacyl-tRNA is channeled during protein synthesis in vivo--i.e., it is directly transferred among the components of the protein-synthesizing machinery and does not mix with aminoacyl-tRNA molecules introduced from outside the cell. To understand the structural basis for these functional properties, we have examined the disposition of aminoacyl-tRNA within the cell. To do this we have developed a Chinese hamster ovary (CHO) permeabilized-cell system using the plant glycoside saponin. We show, using a mixture of free 14C-labeled amino acids and 3H-labeled aminoacyl-tRNAs, that 14C-labeled aminoacyl-tRNAs synthesized endogenously from the free amino acids are preferentially sequestered within the cell, whereas their exogenous 3H counterparts distribute between the inside and outside of the cell based solely on the relative volumes of the two compartments. Furthermore, the endogenous 14C-labeled aminoacyl-tRNA population is resistant to pancreatic ribonuclease action, whereas the 3H molecules are rapidly degraded. We conclude, based on these observations, that aminoacyl-tRNAs synthesized in vivo are continually associated with components of the protein synthesis machinery and are thereby retained within the permeabilized cell and are also protected from RNase action. These data provide independent evidence for the channeling model of protein biosynthesis.
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PMID:A sequestered pool of aminoacyl-tRNA in mammalian cells. 156 55

RNase PH is a Pi-dependent exoribonuclease that can act at the 3' terminus of tRNA precursors in vitro. To obtain information about the function of this enzyme in vivo, the Escherichia coli rph gene encoding RNase PH was interrupted with either a kanamycin resistance or a chloramphenicol resistance cassette and transferred to the chromosome of a variety of RNase-resistant strains. Inactivation of the chromosomal copy of rph eliminated RNase PH activity from extracts and also slowed the growth of many of the strains, particularly ones that already were deficient in RNase T or polynucleotide phosphorylase. Introduction of the rph mutation into a strain already lacking RNases I, II, D, BN, and T resulted in inviability. The rph mutation also had dramatic effects on tRNA metabolism. Using an in vivo suppressor assay we found that elimination of RNase PH greatly decreased the level of su3+ activity in cells deficient in certain of the other RNases. Moreover, in an in vitro tRNA processing system the defect caused by elimination of RNase PH was shown to be the accumulation of a precursor that contained 4-6 additional 3' nucleotides following the -CCA sequence. These data indicate that RNase PH can be an essential enzyme for the processing of tRNA precursors.
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PMID:RNase PH is essential for tRNA processing and viability in RNase-deficient Escherichia coli cells. 164 89

Human immunodeficiency virus (HIV) reverse transcriptase (RT) uses host tRNA(Lys) partially annealed to the primer binding site (PBS) as primer for the initiation of cDNA synthesis. When assaying cDNA synthesis with a template-primer complex formed by an RNA fragment carrying the PBS site and bovine tRNA(Lys) we noticed that an excess of primer tRNA inhibited strongly the DNA polymerase activity of a recombinant HIV RT (p66-p51 heterodimeric form) produced in transformed yeast cells. The same inhibitory effect was observed with animal DNA polymerase alpha, while avian retrovirus RT was neither affected by tRNA(Lys) nor by its specific primer tRNA(Trp). Although the strongest inhibition was observed with tRNA(Lys), other tRNas like tRNA(Phe) and tRNA(Trp) inhibited also the HIV RT, whereas tRNAs specific for valine, proline and glycine had no effect on enzyme activity. Digestion of tRNA(Lys) with pancreatic RNase abolished the inhibition; on the other hand T1 RNase digestion had no effect on the inhibition suggesting a role of the anticodon region in this effect. The 12- and 14-mers corresponding to the anticodon regions of the three bovine tRNA(Lys) isoacceptors inhibited RT activity, indicating that at least an important part of the inhibitory effect could be ascribed to this tRNA region. A strong stimulation of DNA polymerase activity was observed when the effect of tRNA(Lys) was assayed on a recombinant HIV reverse transcriptase produced in a protease deficient yeast strain, which leads to the production of an active p66 enzyme. The same tRNAs that inhibited strongly the heterodimeric form stimulated the p66 form of HIV reverse transcriptase. The results suggest that although both enzymatic forms are able to interact with tRNA(Lys) the topography, as well as the functional implications of the interaction between the precursor and the mature form of HIV reverse transcriptase with the tRNA(Lys) primer, are different.
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PMID:Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA(Lys). 168 23

The region of human angiogenin containing residues 8-21 is highly conserved in angiogenins from four mammalian species but differs substantially from the corresponding region of the homologous protein ribonuclease A (RNase A). Regional mutagenesis has been employed to replace this segment of angiogenin with the corresponding RNase A sequence, and the activities of the resulting covalent angiogenin/RNase hybrid, designated ARH-III, have been examined. The ribonucleolytic activity of ARH-III is unchanged toward most substrates, including tRNA, naked 18S and 28S rRNA, CpA, CpG, UpA, and UpG. In contrast, the capacity of ARH-III to inhibit cell-free protein synthesis is decreased 20-30-fold compared to that of angiogenin. The angiogenic activity of ARH-III is also different; it is actually more potent. It induces a maximal response in the chick chorioallantoic membrane assay at 0.1 ng per egg, a 10-fold lower dose than required for angiogenin. In addition, binding of ARH-III to the placental ribonuclease inhibitor is increased by at least 1 order of magnitude (Ki less than or equal to 7 x 10(-17) M) compared to angiogenin. Thus, mutation of a highly conserved region of angiogenin markedly affects those properties likely involved in its biological function(s); it does not, however, alter ribonucleolytic activity toward most substrates.
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PMID:Replacement of residues 8-22 of angiogenin with 7-21 of RNase A selectively affects protein synthesis inhibition and angiogenesis. 169 38

To determine the essentiality and role of RNase T in RNA metabolism, we constructed an Escherichia coli chromosomal rnt::kan mutation by using gene replacement with a disrupted, plasmid-borne copy of the rnt gene. Cell extracts of a strain with mutations in RNases BN, D, II, and I and an interuppted rnt gene were devoid of RNase T activity, although they retained a low level (less than 10%) of exonucleolytic activity on tRNA-C-C-[14C]A due to two other unidentified RNases. A mutant lacking tRNA nucleotidyltransferase in addition to the aforementioned RNases accumulated only about 5% as much defective tRNA as did RNase T-positive cells, indicating that this RNase is responsible for essentially all tRNA end turnover in E. coli. tRNA from rnt::kan strains displayed a slightly reduced capacity to be aminoacylated, raising the possibility that RNase T may also participate in tRNA processing. Strains devoid of RNase T displayed slower growth rates than did the wild type, and this phenotype was accentuated by the absence of the other exoribonucleases. A strain lacking RNase T and other RNases displayed a normal response to UV irradiation and to the growth of bacteriophages but was severely affected in its ability to recover from a starvation regimen. The data demonstrate that the absence of RNase T affects the normal functioning of E. coli, but it can be compensated for to some degree by the presence of other RNases. Possible roles of RNase T in RNA metabolism are discussed.
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PMID:RNase T affects Escherichia coli growth and recovery from metabolic stress. 170 82

The covalent modification of E. coli arginyl-tRNA synthetase by the 2',3'-dialdehyde derivative of tRNA(Arg) (tRNA(oxArg)) resulted in the complete inactivation of the ATP-PPi exchange and aminoacylation activities of the enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the ArgRS-tRNA(oxArg) covalent complexes indicated that two bands simultaneously appeared on the gel parallel with inactivation corresponding to different higher molecular weights. This result was different from that of the other aminoacyl-tRNA synthetase labeling systems as previously reported. Upon the ribonuclease treatment of the modified ArgRS, less than 15% of both the initial ATP-PPi exchange and aminocylation activities were recovered. During the whole process of labeling and RNase treatment, the two activities of the enzyme were closely associated.
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PMID:Arginyl-tRNA synthetase from Escherichia coli affinity labeling with 3'-oxidized tRNA(Arg). 170 69

Site-directed mutations were introduced in the connecting loops and one of the two stem regions of the RNA pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. The kinetic parameters of valylation for each mutated RNA were determined in a cell-free extract from wheat germ. Structure mapping was performed on most mutants with enzymic probes, like RNase T1, nuclease S1 and cobra venom ribonuclease. An insertion of four A residues in the four-membered connecting loop L1 that crosses the deep groove of the pseudoknot reduces aminoacylation efficiency. Deletions up to three nucleotides do not affect aminoacylation or RNA pseudoknot formation. Deletion of the entire loop abolishes aminoacylation. Although elimination of the pseudoknot is presumed, this could not be demonstrated. Unlike the mutations in loop L1, all mutations in the three-membered connecting loop L2 that crosses the shallow groove of the RNA pseudoknot decrease the aminoacylation efficiency considerably. Nonetheless, the RNA pseudoknot is still present in most mutated RNAs. These results indicate that a number of mutations can be introduced in both loops without abolishing aminoacylation. Results obtained with the introduction of mismatches and A.U base-pairs in stem S1 of the pseudoknot, containing three G.C base-pairs in wild-type RNA, indicate that the pseudoknot is only marginally stable. Our estimation of the gain of free energy due to the pseudoknot formation is at most 2.0 kcal/mol. The pseudoknot structure can, however, be stabilized upon binding the valyl-tRNA synthetase.
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PMID:Mutational analysis of the pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. Aminoacylation efficiency and RNA pseudoknot stability. 173 Oct 70

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

Autoantibody reactive with tRNA was identified by immunoprecipitation of Hela cell extract. Four out of 56 sera from patients with autoimmune chronic active hepatitis (CAH), and four out of 35 sera from patients with primary biliary cirrhosis (PBC) contained antibody directed against gel-purified tRNA in Hela cell extract, but no sera obtained from CAH type B, CAH non-A, non-B, or healthy volunteers did. Further studies on these eight anti-tRNA sera disclosed that 6 of the 8 sera that immunoprecipitated tRNA from Hela cell extract, reacted with purified tRNA, but reacted with neither 5sRNA nor ribosomal RNA species. After proteinase and deoxyribonuclease digestion of Hela cell extract, the epitope for these 6 sera was conserved, and the antigen was sensitive to ribonuclease (anti-tRNA serum). Purified Hela cell DNA digested with Eco RI or Hind III (denatured or non-denatured) could not be immunoprecipitated by these sera. In a patient with autoimmune CAH, the anti-tRNA antibody was weakly positive at week 2 and disappeared 2 months after steroid therapy started, "in parallel" with disappearance of anti-nuclear antibody. In the other 2 sera, the antigen was sensitive to proteinase.
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PMID:Autoantibody specific for transfer ribonucleic acid (tRNA) in patients with autoimmune chronic active hepatitis and primary biliary cirrhosis. 177 87

A spermidine-dependent endoribonuclease (designated as RNase 65) activity requires both RNA and protein components (Nashimoto et al. (1991) Biochem. Biophs. Res. Comm. 176:1163-1169). In this study, we fractionated RNAs from mouse FM3A cell extracts and showed that an RNA fraction containing two major RNAs and two minor ones restored the micrococcal nuclease-inhibited RNase 65 activity. Partial sequences of these four RNA species were determined by chemical RNA sequencing. A sequence homology search revealed that the two major RNAs were glutamine tRNA lacking its 3' terminus, and that the two minor RNAs were initiator methionine tRNA and glycine tRNA lacking their 3' termini.
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PMID:Transfer RNA lacking its 3' terminus is required for spermidine-dependent ribonuclease 65 activity in mouse FM3A cell extracts. 187 44


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