Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The relative affinities of all Escherichia coli amino-acyl-tRNAs for E. coli elongation factor (EF) Tu-GTP have been measured by two independent applications of the competition form of the ribonuclease resistance assay. The set of aminoacyl-tRNAs includes at least one tRNA for each of the 20 amino acids as well as purified isoacceptor tRNA species for arginine, glycine, leucine, lysine, and tyrosine. In the first competition study, [3H]Phe-tRNA was used as the competing aminoacyl-tRNA against [14C]aminoacyl-tRNA in the set of all tRNAs; in the second study, [3H]Leu-tRNALeu4 was used as the competing aminoacyl-tRNA. The relative order of aminoacyl-tRNA affinities for EF-Tu-GTP was the same in each study. The results indicate that the affinity of EF-Tu-GTP at 4 degrees C, pH 7.4, is strongest for Gln-tRNA and weakest for Val-tRNA. Both Gly-tRNA and Pro-tRNA bind very strongly to EF-Tu-GTP relative to other aminoacyl-tRNAs. Various models of ternary complex interactions are discussed in light of the new data. Although the properties of the amino acid substituent are primarily responsible for the differences in relative affinities among the noninitiator aminoacyl-tRNAs, the results for the four isoacceptor species of Leu-tRNALeu indicate that the secondary structural features of the tRNA are also influential.
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PMID:Relative affinities of all Escherichia coli aminoacyl-tRNAs for elongation factor Tu-GTP. 637 Sep 98

The addition of detergent-solubilized influenza C virus to a reaction mixture containing ATP, CTP, GTP, [3H]UTP, and Mg2+ leads to the synthesis of an acid-insoluble, radioactive product, which is ribonuclease-sensitive. The dinucleoside monophosphate ApG clearly enhances the reaction rate, a fact which indicates that influenza C viruses follow the same strategy of transcription as influenza A and B viruses, the other members of the orthomyxovirus family.
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PMID:Influenza-C-virion-associated RNA-dependent RNA-polymerase activity. 654 51

Escherichia coli elongation factor (EF-Tu) binds aminoacyl-tRNAs (aa-tRNA) not only in the presence of GTP but also in the presence of GDP. Complex formation leads to a protection of the aa-tRNA against nonenzymatic deacylation and digestion by pancreatic ribonuclease, as well as to a protection of EF-Tu against proteolysis by trypsin. The equilibrium constant for the binding of Phe-tRNAPheyeast for example to EF-Tu.GDP has been determined to be 0.7 X 10(5) M-1 which is 2 orders of magnitude lower than the equilibrium constant for Phe-tRNAPheyeast binding to EF-Tu.GTP. In the presence of kirromycin, aminoacyl-tRNA binding to EF-Tu.GDP is not affected as much: Phe-tRNAPheyeast is bound with an equilibrium constant of 3 X 10(5) M-1. While there is also a measurable interaction between EF-Tu.GTP and tRNA, such an interaction cannot be detected with EF-Tu.GDP and tRNA, not even at millimolar concentrations. A so far undetected complex formation between aminoacyl-tRNA and EF-Tu.GTP in the presence of pulvomycin, however, could be detected. The results are discussed in terms of the structural requirements of ternary complex formation and in the light of proofreading schemes involving A-site binding on the E. coli ribosome.
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PMID:The elongation factor Tu binds aminoacyl-tRNA in the presence of GDP. 674 37

The complex of elongation factor Tu with GTP (EF-Tu.GTP) reacts with N or epsilon -bromoacetyl-lys-tRNA ( or epsilon BrAcLys-tRNA) to form a functional covalently linked complex (XLTC). The site of cross-linking must be near the site on EF-Tu.GTP that binds the aminoacyl moiety of aminoacyl transfer ribonucleic acid (AA-tRNA). For identification of this site, a nanomole of purified XLTC prepared from or epsilon BrAc[(14)C]Lys-tRNA was digested first with RNase A and then with trypsin, and the peptides were resolved by high-performance liquid chromatography using a c8 reverse-phase column. A single peptide contained 80% of the label. The amino acid composition of this peptide was identical with that of residues 59-74 in EF-Tu. The NH2-terminal sequence of the peptide was determined to be Fly-Ile-Thr-Ile, which are residues 59-62 in EF-Tu. The modified amino acid was identified as pi - (carboxymethyl)histidine, which establishes that His-66 is at or near the AA-tRNA binding site on EF-Tu.GTP.
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PMID:Identification of a histidine residue near the aminoacyl transfer ribonucleic acid binding site of elongation factor Tu. 691 6

Jekowsky et al. reported recently that elongation factor Tu:GTP complex from Escherichia coli protected aminoacyl-tRNA from digestion by pancreatic RNase (I). On the basis of their finding, we have developed the "RNase-resistance assay" for determination of the dissociation constant of aminoacyl-tRNA from aminoacyl-tRNA:EF-Tu:GTP complex. By the use of this sensitive assay, the dissociation constants were estimated to be 3.6 x 10(-7) M for Ala-tRNA1Ala (Torulopsis utilis), 7.9 x 10(-8) M for Phe-tRNAPhe (Escherichia coli), 8.1 x 10(-7) M for initiator Met-tRNAfMet (Escherichia coli), and 5.4 x 10(-6) M for Gly-tRNA1Gly (Staphylococcus epidermidis) participating in cell wall biosynthesis. Moreover, using a relatively large amount of EF-Tu:GTP, we have been able to detect the ternary complexes of initiator Met-tRNAfMet and Gly-tRNA1Gly with EF-Tu:GTP even by the method of gel filtration.
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PMID:Interaction of initiator Met-tRNArMet (Escherichia coli) and Gly-tRNAIGly (Staphylococcus epidermidis) with bacterial elongation factor Tu:GTP complex. 702 61

When EF-Tu was photooxidized for 20 min at 0 degrees C in the presence of 10 microM GDP and 5 microM rose bengal, the activity to promote the binding of [14C]Phe-tRNA to ribosomes was rapidly lost, while the activity to bind [3H]GDP remained intact. The activity of EF-Tu to interact with Phe-tRNA and ribosomes, as assessed by protection of [14C]Phe-tRNA against RNase A digestion and by methanol-induced uncoupled GTPase activity, respectively, was also inactivated under the above conditions. It was found, however, that these activities were fully protected in the presence of aminoacyl-tRNA and GTP, indicating that the active site(s) of EF-Tu for interaction with aminoacyl-tRNA and ribosomes could be protected against photooxidation in the ternary aminoacyl-tRNA . EF-Tu . GTP complex. Comparison of the amino acid composition of EF-Tu photooxidized in the form of EF-Tu . GDP with that of the intact EF-Tu revealed that only 1.4 residues of histidine were damaged. On the other hand, no histidine residue was lost when EF-Tu was oxidized in the presence of both aminoacyl-tRNA and GTP. The photooxidized EF-Tu . GDP was then partially degraded with trypsin and each of the resulting tryptic fragments, D, B, and C (Arai, Nakamura, Arai, Kawakita, and Kaziro (1976) J. Biochem. 79, 69-83), was analyzed for histidine content. The results indicated that fragments B, C, and D had lost 0.7, 0.5, and 0.2 residues of histidine, respectively. Since fragment B contains the cysteine residue which is essential for interaction with aminoacyl-tRNA and ribosomes, the above results suggest that a histidine residue in fragment B may also play an essential role in the interaction with aminoacyl-tRNA and ribosomes.
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PMID:Selective photooxidation of histidine residues in polypeptide chain elongation factor Tu from E. coli. 703 Oct 46

Dissociation constant of aminoacyl-tRNA:EF-Tu:GTP complex into aminoacyl-tRNA and EF-Tu:GTP was estimated by the RNase-resistance assay developed by us. The experimental results showed that EF-Tu:GTP has a high affinity for Met-tRNAfMet (E. coli) and Met-tRNAmMet, but not fMet-tRNAfMet. The process of the formylation for Metm-tRNAfMet may provide a security against incorrect translation at GUG (valine) and UUG (leucine) codons in the elongation step.
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PMID:Interaction of fMet-tRNAfMet, Met-tRNAfMet, and Met-tRNAmMet with bacterial elongation factor Tu:GTP complex: discrimination against fMet-tRNAfMet. 703 10

The present investigation was undertaken to see to what extent the alpha-amino group of the amino acid, the side chain of the amino acid of aminoacyl-tRNA, and the tRNA structure are involved in determining the affinity of aminoacyl-tRNA for bacterial elongation factor Tu-GTP complex. Various aminoacyl-tRNAs, mis-aminoacylated tRNAs, and formylated aminoacyl-tRNAs were prepared, and the dissociation constants of the ternary complexes of aminoacyl-tRNA with ET-Tu: GTP were determined by the RNase-resistance assay. The results indicated that the free amino-acid group of the amino acids in aminoacyl-tRNA is strongly required for binding with EF-Tu : GTP. In this concentration, the biological significance of formylation for Met-tRNAMetf species is discussed.
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PMID:Interaction of aminoacyl-tRNA with bacterial elongation factor Tu: GTP complex: effects of the amino group of amino acid esterified to tRNA, the amino acid side chain, and tRNA structure. 704 Mar 60

Isolated nuclei incubated with [14C]protein hydrolysate are shown to incorporate labelled amino acids into the acid-insoluble fraction. Purified chromatin and the complex of DNA with firmly bound proteins possess similar ability. The optimum pH of the reaction is 6.5-7.0, 2 mM MgCl2 stimulates incorporation, the temperature optimum is 37-40 degrees C. Chloramphenicol depresses incorporation by 70%, puromycin by 40%, cycloheximide does not affect the chromatin activity. Incorporation does not depend on the presence of ATP or GTP, and is substantially inhibited by deoxyribonuclease but not by ribonuclease treatment of chromatin or of the nuclei. Specific activity of firmly bound chromatin non-histone proteins is higher than that of labile bound ones; histones are not labelled. After pronase treatment of proteins radioactivity changes to an acid-soluble state. The molecular weight of isolated labelled polypeptides is about 6000 as shown by gel filtration and the analysis of NH2-terminal amino acids. Labelled polypeptides firmly bound to DNA consist of 7-10 amino acids. Specific activity of proteins firmly bound to DNA increases linearly with the time of incubation of chromatin with [14C]protein hydrolysate, the activity curve of labile bound non-histone proteins has a distinct sygmoid character. The polypeptide-synthesizing activity of rat liver chromatin increases between 9 h and 21 h after partial hepatectomy. Irradiation with 800 rads 30 min before the operation prevents activation of amino acid incorporation. From nine amino acids studied alanine, methionine, lysine, tyrosine and arginine are not incorporated in the system described. Glutamic acid is polymerized most effectively. Glutamine, asparagine and glycine are incorporated 7-8 times less. The data are given indicating that the incorporation is not random when an amino acid mixture is present. Preincubation of chromatin with NAD+ but not with its analogues increases the polypeptide-synthesizing activity of chromatin. The activation is prevented by thymidine and nicotinamide. Storage (18 h at 2-4 degrees C) brings about a complete loss of the polypeptide-synthesizing activity of chromatin. The ability of 'old' chromatin to incorporate amino acids can be restored by preincubating it with NAD+. Storage of chromatin in the presence of 5 mM adenosine 3',5'-monophosphate (cAMP) does not result in decrease of the polypeptide-synthesizing activity. It is assumed that poly-(ADP-ribose) is the energy source for amino acid activation in the system described.
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PMID:Polypeptide-synthesizing activity of eukaryotic chromatin. Properties, dependence on poly(ADP-ribose) and connection with the cell cycle. 737 37

The higher order structure of the functionally important 530 loop in Escherichia coli 16S rRNA was studied in mutants with single base changes at position 517, which significantly impair translational fidelity. The 530 loop has been proposed to interact with the EF-Tu-GTP-aatRNA ternary complex during decoding. The reactivity at G530, U531 and A532 to the chemical probes kethoxal, CMCT and DMS respectively was increased in the mutant 16S rRNA compared with the wild-type, suggesting a more open 530 loop structure in the mutant ribosomes. This was supported by oligonucleotide binding experiments in which probes complementary to positions 520-526 and 527-533, but not control probes, showed increased binding to the 517C mutant 70S ribosomes compared with the non-mutant control. Furthermore, enzymatic digestion of 70S ribosomes with RNase T1, specific for single-stranded RNA, substantially cleaved both wild-type and mutant rRNAs between G524 and C525, two of the nucleotides involved in the 530 loop pseudoknot. This site was also cleaved in the 517C mutant, but not wild-type rRNA, by RNase V1. Such a result is still consistent with a more open 530 loop structure in the mutant ribosomes, since RNase V1 can cut at appropriately stacked single-stranded regions of RNA. Together these data indicate that the 517C mutant rRNA has a rather extensively unfolded 530 loop structure. Less extensive structural changes were found in mutants 517A and 517U, which caused less misreading. A correlation between the structural changes in the 530 loop and impaired translational accuracy is proposed.
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PMID:Structural changes in the 530 loop of Escherichia coli 16S rRNA in mutants with impaired translational fidelity. 756 70


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