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 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

The involvement of each of the amino acid residues of the I-Ak-restricted T cell determinant RNase(43-56) was examined in detail using a series of peptides containing single amino acid substitutions. Four positions were identified as being essential for the formation of the determinant, Phe-46, Val-47, His-48, and Leu-51. When these four residues were substituted into the backbone of the unrelated peptide HA(130-144), a nonstimulatory peptide was obtained. The inclusion of an additional residue, Val-54, resulted in a chimeric peptide, RN/HA2, which was nearly as active as the native molecule. The peptide RN/HA2 was able to prime in vivo for RNase reactivity, confirming that these five residues contained all of the specificity of the RNase(43-56) determinant. The role of three of these critical residues was examined using both a functional competition assay and an in vivo priming assay. It was ascertained that the Phe-46 was directly involved in contacting the TCR, while the His-48 and Leu-51 were either involved in binding to the I-Ak molecule or in determining the conformation of the peptide. Thus, by critically evaluating the contribution of each of the amino acid residues in a T cell determinant, we were able to generate a chimeric peptide only containing 5 of 15 residues from the RNase(43-56) sequence that was functionally identical to the native RNase(43-56) molecule both in vitro and in vivo.
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PMID:Reconstruction of the immunogenic peptide RNase(43-56) by identification and transfer of the critical residues into an unrelated peptide backbone. 247 59

To make strong statements about possible tertiary structure or the relative stability of regions of secondary structure, the structure-probing experiments must go further than single-hit reactions. Some elements of the environment of the RNA molecule must be altered systematically. Knowledge of the effects of ions or other interacting factors on the activity or physical parameters (e.g., NMR and melting cooperativity) of the RNA help in experimental design. For example, the copious work on tRNA(Phe) compared the crystal and solution structures and allowed the direct correlation of Mg2+ stabilization of the tertiary structure of that molecule. Figure 3 demonstrates that pre-tRNA(Leu-3) responds to Mg2+ depletion in the same manner as detected by the appearance of highly sensitive RNase cleavage sites in the D and T psi C loops. Similar experiments titrating polyamine concentrations suggested that secondary structure was more efficiently stabilized by polyamines than by Mg2+. The variation of Mg2+ concentrations has been used to gain additional information about other RNA structures. Others have used protein-RNA interactions to approach the question of the functional structure of a RNA (for examples, see Ref. 3). Thus, the ideal parameters to choose would be those known to affect the function of the RNA. The variation of Mg2+ and polyamine concentrations would minimally suggest regions of greater or lesser secondary or tertiary structure stability.
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PMID:Enzymatic approaches to probing of RNA secondary and tertiary structure. 248 14

A simple and precise method was developed for the separation of nucleosides including modified nucleosides and oligonucleotides. Nineteen kinds of nucleosides were completely separated by HPLC using an ODS column (TSK-gel ODS 80TM) and aqueous mobile phases. The RNA molecule was digested by base restrictive RNase (RNase A, RNase T1) and the digests were separated chromatographically into each oligonucleotide. The nucleoside composition of an oligonucleotide was then determined by this analytical system. It is thus possible to fit the oligonucleotide in the original RNA molecule by using modified bases as markers. The reaction site of quinacrine mustard for tRNA(Phe) (from yeast) could be determined by this analytical system.
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PMID:High resolution chromatography of ribonucleosides and its application to RNA analysis. 248 88

The adk genes from several thermosensitive (ts) mutants of Escherichia coli were cloned and sequenced. The mutations responsible for the thermolability of the gene product, the enzyme adenylate kinase, were established. From five independently isolated strains analysed, two contain a CCG to TCG transition changing proline 87 to serine (P87S), another two have a TCT to TTT transition that mutates serine 129 to phenylalanine (S129F), and the last one was found not to contain a mutation in the adk gene. Overproducing strains were constructed that contain ts genes in the genome as well as in the plasmids. These strains grow at high temperature, although much slower than wild-type. Most probably, the high rate of synthesis of adenylate kinase compensates for the destruction of the thermolabile protein by the elevated temperature. Mutated proteins were purified. The P87S but not the S129F mutation was found to cause thermosensitivity of the adenylate kinase reaction. Revertants of thermosensitivity were isolated and the nature of the mutation was determined by the RNase digestion method of RNA-DNA hybrids and by DNA sequencing. The revertants of the P87S mutation regained the wild-type sequence, whereas the revertants of the S129F strain retained the original mutation in the adenylate kinase gene. These results are discussed in the light of the three-dimensional structure of the enzyme and the possible role of adenylate kinase in phospholipid synthesis.
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PMID:Adenylate kinases from thermosensitive Escherichia coli strains. 254 33

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

Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in the biosynthesis from phenylalanine of a wide variety of phenylpropanoid natural products including lignin, flavonoid pigments, and phytoalexins. In bean (Phaseolus vulgaris L.), PAL is encoded by a family of three genes. We show here by RNase protection with gene-specific probes that these genes are expressed differentially during development and in response to different environmental cues. While all three genes are expressed at high levels in roots, only PAL1 and PAL2 are expressed in shoots and only PAL1 is expressed in leaves. Strikingly, PAL2 is expressed at very high levels in petals, where PAL1 is only very weakly expressed and PAL3 is not expressed. All three genes are induced by mechanical wounding of hypocotyls, but fungal infection only activates PAL1 and PAL3. Illumination of etiolated hypocotyls activates PAL1 and PAL2 but not PAL3. Corresponding differential patterns of synthesis of specific PAL polypeptide isoforms were observed by two-dimensional gel electrophoretic analysis of in vitro translation products encoded by RNA isolated from hypocotyls stimulated by light, wounding, or infection. The specific isoforms encoded by transcripts of the three PAL genes were identified by inhibition of synthesis in vitro with gene-specific anti-sense transcripts followed by comparative two-dimensional gel electrophoretic analysis of the pattern of translation products. These data indicate that selective expression of PAL genes encoding functional variants is governed by a complex set of regulatory networks for developmental and environmental control of phenylpropanoid biosynthesis.
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PMID:Differential regulation of phenylalanine ammonia-lyase genes during plant development and by environmental cues. 276 71

We have determined the nucleotide sequence changes caused by three missense mutations leading to the production of inactive colicin E3 proteins. The ceaC1 mutation, affecting the translocation of colicin E3 through bacterial membranes, is caused by a serine to phenylalanine change at position 37 within the glycine-rich region at the N-terminal part of colicin E3. This confirms previous results suggesting a role for this domain in colicin uptake by sensitive cells. The ceaC2 and ceaC3 mutations, abolishing colicin E3 RNase activity, affect the C-terminal enzymatic domain of the molecule. In the ceaC2 mutant, serine at position 529 was converted to leucine. The ceaC3 mutation replaced a glycine residue at position 524 with an aspartic acid residue. The two mutations ceaC2 and ceaC3 yield information on the amino acid residues involved in the RNase activity of colicin E3.
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PMID:DNA sequence analysis of three missense mutations affecting colicin E3 bactericidal activity. 283 30

The amino acid sequence and disulfide bond pairing of human tumor derived angiogenin, the first tumor angiogenesis factor to be isolated in pure form from human sources, have been determined by conventional sequencing techniques adapted and applied to nanomole and subnanomole levels of material. Angiogenin, obtained from conditioned media of a human colonic adenocarcinoma cell line, is a single-chain protein consisting of 123 amino acids with the following sequences: less than Glu1-Asp-Asn-Ser-Arg-Tyr-Thr-His- Phe-Leu-Thr-Gln-His-Tyr-Asp15-Ala-Lys-Pro-Gln-Gly-Arg-Asp-Asp- Arg-Tyr-Cys-Glu-Ser-Ile-Met30- Arg-Arg-Arg-Gly-Leu-Thr-Ser-Pro-Cys-Lys-Asp-Ile-Asn-Thr- Phe45-Ile-His-Gly-Asn-Lys-Arg-Ser -Ile-Lys-Ala-Ile-Cys-Glu-Asn-Lys60-Asn-Gly-Asn-Pro-His-Arg-Glu-Asn -Leu-Arg-Ile -Ser-Lys-Ser-Ser75 -Phe-Gln-Val-Thr-Thr-Cys-Lys-Leu-His-Gly-Gly-Ser-Pro-Trp-Pro90-Pro -Cys-Gln-Tyr -Arg-Ala-Thr-Ala -Gly-Phe-Arg-Asn-Val-Val-Val105-Ala-Cys-Glu-Asn-Gly-Leu-Pro-Val- His-Leu-Asp-Gln-Ser-Ile-Phe120-Arg-Arg-Pro123-OH. Three disulfide bonds link the half-cystinyl residues 26-81, 39-92, and 57-107. The sequence is homologous to that of the pancreatic ribonucleases with 35% identity and many of the remaining residues conservatively replaced. Similarities are especially apparent around the major active-site residues His-12, Lys-41, and His-119 of ribonuclease which are conserved as are three of the four disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Amino acid sequence of human tumor derived angiogenin. 286 94

Pregnant rats were loaded with L-phenylalanine, and the distributions of [14C]leucine and [14C]urea into fetal plasma and tissues were examined. Uptake of [14C]leucine into the supernatant and protein fractions of fetal plasma and tissues was low in the rats loaded with phenylalanine. In contrast, [14C]urea was distributed identically in both groups, indicating that maternal hyperphenylalaninemia did not affect blood flow across the placenta. Administration of phenylalanine and p-chlorophenylalanine produced amino acid imbalance in fetal tissues. Along with these changes, polysomes of the affected fetal heart and brain disaggregated without changes in the ribonuclease activity. These results indicate that high phenylalanine levels in maternal plasma disturb the active transport of amino acids across the placenta, causing an amino acid imbalance and disaggregation of polysomes in fetal heart and brain. These changes may contribute to the congenital heart disease and mental retardation of maternal phenylketonuria.
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PMID:Effects of phenylalanine loading on protein synthesis in the fetal heart and brain of rat: an experimental approach to maternal phenylketonuria. 294 18


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