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)

The tRNA nucleotidyltransferase activity (3H-CMP incorporation into 3'-terminus of tRNApC) in cytoplasmic fractions of various types of cells such as Ehrlich ascites tumor cells, mouse liver and spleen cells, rat spleen, lymph node, and macrophages cells was found to be dependent on the concentrations of nucleoside 5'-triphosphates (ATP, GTP, UTP, dATP, dGTP, dCTP, and/or dTTP). The purified tRNA nucleotidyltransferase did not show such dependency. The dependency of the enzyme activity on nucleoside 5'triphosphates in the crude cytoplasmic fractions was possibly due to the presence of inhibitors which interfere with the repair system of defective 3'-termini of tRNA. Two kinds of inhibitors were distinguishable in the cytoplasmic fractions. One was unstable on heat treatment at 55 decrees C and showed ribonuclease activity for the tRNA 3'-terminus. The other which lacked ribonuclease activity was rather stable to the heat treatment and inhibited purified tRNA nucleotidyltransferase. The actions of both inhibitors were suppressed by nucleoside 5'-triphosphates.
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PMID:Effect of nucleoside 5'-triphosphates on tRNA nucleotidyltransferase activity in cytoplasmic fractions of various types of mammalian cells. 42 63

Escherichia coli contains multiple exoribonucleases. Strains lacking the exoribonucleases RNase II, D, BN, T, and PH are inviable. The introduction of a chromosomal, wild-type copy of the gene for any one of these enzymes is sufficient to allow cell growth, with the enzymes being in the following order of effectiveness: RNase T > RNase PH > RNase D > RNase II > RNase BN. The data indicate that these five exoribonucleases functionally overlap in vivo and that any one of them can take over the functions of all the others, although with various efficiencies.
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PMID:The presence of only one of five exoribonucleases is sufficient to support the growth of Escherichia coli. 140 Feb 19

The pyrE gene, encoding the pyrimidine biosynthetic enzyme orotate phosphoribosyltransferase, is the promoter distal gene of the dicistronic orfE-pyrE operon. The promoter proximal orfE gene, whose transcription and translation is important for regulation of the pyrE attenuator, encodes a 238-amino acid residue protein which was recently identified as the phosphorolytic ribonuclease, RNase PH, that removes nucleotides from the 3' ends of tRNA precursors. In this paper we report the construction of a plasmid, which overexpresses the orfE and pyrE gene products substantially, as well as the purification of the OrfE protein by ammonium sulfate precipitation and chromatography on phosphocellulose. The highly purified protein catalyzes the phosphorolytic cleavage of poly(A) at a rate of 1.6 mumol/min/mg and the formation of CDP from tRNA-CCA-Cn and orthophosphate at a rate equal to 0.14 mumol/min/mg, as characteristic for RNase PH. OrfE/RNase PH contains helix-turn-helix motifs resembling those in DNA-binding proteins, and it binds nonspecifically to DNA. On SDS gels, OrfE/RNase PH migrates as two distinct protein bands. This heterogeneity might be caused by post-translational modification other than proteolysis, or may be an electrophoretic artifact. The native protein is composed of two or more subunits.
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PMID:Overexpression and rapid purification of the orfE/rph gene product, RNase PH of Escherichia coli. 151 52

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

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

RNase PH from extracts of Escherichia coli was purified to homogeneity and subjected to NH2-terminal sequencing. Comparison of this sequence with all open reading frames in the GenBank data base revealed at least 95% identity to an unidentified open reading frame (orfE) upstream of pyrE at 81.7 min on the E. coli chromosome. Clones of orfE overexpress RNase PH activity, verifying that orfE encodes this ribonuclease. We suggest that orfE be renamed rph.
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PMID:Escherichia coli orfE (upstream of pyrE) encodes RNase PH. 188 37

A mutant strain deficient in RNase T was isolated and used to study the role of this enzyme in Escherichia coli. Strains lacking as much as 70% of RNase T activity, alone or in combination with the absence of other RNases, display normal growth properties. However, in cca strains, which lack tRNA nucleotidyltransferase, RNase T-deficient derivatives accumulate lower levels of defective tRNA and grow at increased rates compared to their RNase T+ parents. Slow-growing cca strains revert to a faster-growing form that contains less defective tRNA but which is still cca. All of these strains have decreased levels of RNase T. These data indicate that RNase T is responsible for nucleotide removal during the tRNA end-turnover process and that the amount of defective tRNA in cells is determined by the relative levels of RNase T and tRNA nucleotidyltransferase.
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PMID:RNase T is responsible for the end-turnover of tRNA in Escherichia coli. 241 40

Final trimming of the 3' terminus of tRNA precursors in Escherichia coli is thought to proceed by an exonucleolytic mechanism. However, mutant strains lacking as many as four exoribonucleases known to act on tRNA still grow normally and process tRNA normally. Extracts from such a multiple-RNase-deficient strain accurately mature tRNA precursors exonucleolytically in vitro in a reaction that requires inorganic phosphate. Here we show that this reaction is not due to polynucleotide phosphorylase (PNPase) but, rather, that it is mediated by a phosphate-requiring exonuclease that we have named RNase PH. Purified PNPase is incapable of completely processing tRNA precursors, and extracts from a PNPase- strain retain full activity for phosphorolytic processing. Although both PNPase and RNase PH act in a phosphorolytic manner, they differ substantially in size and substrate specificity. RNase PH has a molecular mass of 45-50 kDa and favors tRNA precursors as substrates. The possible physiological role of RNase PH and the advantages of phosphorolytic processing are discussed.
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PMID:RNase PH: an Escherichia coli phosphate-dependent nuclease distinct from polynucleotide phosphorylase. 245 97

The role of tRNA nucleotidyltransferase in Escherichia coli has been uncertain because all tRNA genes studied in this organism already encode the -C-C-A sequence. Examination of a cca mutant, originally thought to contain 1-2% enzyme activity, indicated that it actually produces an inactive fragment of 40 kd compared to 47 kd for the wild-type enzyme due to a nonsense mutation in its cca gene. To confirm that the residual activity in extracts of this strain is due to another enzyme, and that tRNA nucleotidyltransferase is non-essential, we have interrupted the cca gene in vitro, and transferred this mutant gene to a variety of strains. In all cases mutant strains are viable, although as much as 15% of the tRNA population contains defective 3' termini, and no tRNA nucleotidyltransferase is detectable. Mutant strains grow slowly, but can be restored to more normal growth by a relA mutation or by a decrease in RNase T activity. In the latter case the amount of defective tRNA decreases dramatically. These findings indicate that tRNA nucleotidyltransferase is not essential for E. coli viability, and therefore, that all essential tRNA genes in this organism encode the -C-C-A sequence.
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PMID:tRNA nucleotidyltransferase is not essential for Escherichia coli viability. 331 29

1. Different reaction steps involved in protein synthesis were studied in skeletal muscles from control and myopathic hamsters. 2. There was no difference between partially purified aminoacyl-tRNA synthetases from myopathic and control animals in yield or catalytic activity, as tested with exogenous deacylated tRNA. 3. However, isolated deacylated tRNA from myopathic muscle was aminoacylated by these synthetases to a lesser extent than that derived from control muscle. 4. Addition of deacylated tRNA isolated from control muscle improved the performance of pH5 enzymes from myopathic muscle in polypeptide synthesis on homologous polyribosomes; tRNA isolated from myopathic animals did not. 5. Preparation of extracts from both types of animals in the presence of the ribonuclease-absorbent bentonite led to an increased capacity of endogenous tRNA to accept amino acids in pH5 enzymes prepared from normal and abnormal tissue, but the difference between the two systems remained the same. 6. Total tRNA nucleotidyltransferase activity, tested with twice-pyrophosphorolysed rat liver tRNA, was identical in both extracts. 7. Added tRNA nucleotidyltransferase incorporated more AMP and CMP into endogenous tRNA with the pH5 enzyme from myopathic muscle than with that from control muscle. 8. Preincubation of deacylated tRNA from myopathic muscle with ATP, CTP and tRNA nucleotidyltransferase more than doubled its subsequent aminoacyl-acceptor activity, and halved the extent of the defect relative to aminoacylation of control tRNA similarly treated. Endogenous tRNA in pH5 enzyme preparations behaved likewise. 9. It is suggested that a 3'-exonuclease in myopathic muscles attacks tRNA molecules in such a way that some of them remain substrates for tRNA nucleotidyltransferase, which may incorporate into RNA not only AMP and CMP, but also GMP. 10. Cell-free protein synthesis in preparations from myopathic hamster muscles is limited by the supply of intact tRNA molecules.
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PMID:Evidence for defective transfer ribonucleic acid in polymyopathic hamsters and its inhibitory effect on protein synthesis. 472 37


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