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:3.1.27.3 (RNase T1)
1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A second major species of leucine tRNA, tRNA Leu UAG (formerly designated tRNA Leu CUA) was purified from baker's yeast in a three-step procedure entailing BD-cellulose chromatography in the presence and absence of Mg2+ and Sephadex G-100 gel filtration. Results of aminoacylation and partial RNase T1 digestion experiments showed that this tRNA retains a native conformation under conditions that denature yeast tRNA Leu m5CAA (tRNA3 Leu). The primary structure of baker's yeast tRNA Leu UAG was elucidated by application of sensitive radioactive isotope derivative ("postlabeling") methods. Complete RNase T1 and A and partial RNase U2 fragments, prepared from non-radioactive tRNA and 5'-half and 3'-half molecules, were separated by two-dimensional polyethyleneimine-cellulose anion-exchange thin-layer chromatography and isolated by a novel micropreparative procedure affording high yields of these compounds in sufficient purity for subsequent tritium derivative analysis. Base composition and sequence of oligonucleotides were analyzed by tritium derivative methods. Molar ratios of the fragments were determined from the radioactivity of 3H-labeled nucleoside trialcohols in combination with base analysis. 2'-O-Methylated guanosine was characterized using the [gamma-32P]ATP/polynucleotide kinase reaction. The analysis of classical complete and partial RNase digests by the tritium derivative methods yielded the complete nucleotide sequence of the tRNA. A total of about 20 A260 units of the RNA was used for analysis, i.e. considerably less material than required for conventional spectrophotometric analysis. A different sequencing approach, consisting of a combination of "readout sequencing" with tritium sequencing of complete RNase T1 and A fragments, was applied to the 3'-half molecule. The 3'-half molecule was labeled with 32P at its 5' terminus, partially degraded with RNase T1, U2, and Phy1 and with alkali, and subjected to polyacrylamide gel electrophoresis. The sequence was read off the gel on the basis of cleavage patterns and size of the fragments. While the readout procedure provided only the positions of A, U, C, and G residues in the chain, additional information from tritium derivative analysis was utilized to define the positions of the modified nucleosides. The readout sequencing procedure was found to require less than 0.01 A260 unit of RNA and the analysis of the complete fragments about 6 A260 units. Interesting structural features of tRNA Leu UAG are (a) the location of unique, leucine tRNA iso-acceptor-specific sequences next to U-8, a constant nucleotide participating in synthetase recognition, (b) the occurrence of 1-methyladenosine in the T loop, a modification not present in the structurally related tRNA Leu m5CAA, and (c) the unusual presence of an unmodified uridine in the first position of the anticodon, which may be related to the unusual coding properties reported for this tRNA.
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PMID:Yeast tRNA Leu UAG. Purification, properties and determination of the nucleotide sequence by radioactive derivative methods. 37 75

2-Thiocytidine 5'-triphosphate, s2CTP, is able to replace CTP as a substrate for tRNA nucleotidyltransferase. s2CMP can be incorporated into both cytidine sites of the C-C-A terminus common to all tRNAs, and in the absence of ATP into at least two additional positions. This was shown by alkylation of the 2-thiocytidine residues with iodo[14C]acetamide, total nucleoside analysis, microgel electrophoresis and analysis of RNase T1 fragments of these tRNAs. The incorporation of the 3'-terminal AMP is not influenced by the additional s2CMP residues at pH 9.0. However, at pH 7.6 the additional s2CMP residues are hydrolysed and AMP can be incorporated into the normal position. Two different tRNAs with terminal 2-thiocytidine alkylated by iodoacetamide inhibit tRNA nucleotidyltransferase. This inhibition is significantly slower if an elongated species is used compared to a tRNA with alkylated 2-thiocytidine in the normal position 75. The addition of 2-mercaptoethanol reactivates the enzyme and leads to a cytidine containing tRNA. This reaction identifies the attacking nucleophile of the enzyme as cysteine residue, which is probably identical to a cysteine residue found in a similar experiment reported previously. The mechanism of the enzymatic and chemical reactions is discussed.
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PMID:Reversible inactivation of tRNA nucleotidyltransferase from baker's yeast by tRNAPhe containing iodoacetamide-alkylated 2-thiocytidine in normal and additional positions. 37 62

A modification of the known method for obtaining radioactive fingerprints from non-radioactive nucleic acids by labelling a digest with 5'-hydroxyl polynucleotide kinase and [gamma-32P]-ATP has been applied to RNase T1 digests from various high molecular weight virus RNAs and to ovalbumin mRNA. Fractionation of the resultant [32P]-labelled T1 RNase digests by two-dimensional polyacrylamide electrophoresis demonstrates that in the case of virus RNAs, the fingerprints thus obtained are very similar to those derived from uniformly labelled RNAs. The value of this technique is that it requires only 1-5 microgram of purified virus RNA and at least three orders of magnitude less radioactivity than is routinely employed in preparing uniformly labelled RNA.
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PMID:Oligonucleotide mapping of non-radioactive virus and messenger RNAs. 41 3

Potato spindle tuber viroid (PSTV), a small infectios RNA, has been completely digested with RNase T1 and RNase A, and the resulting oligonucleotides have been sequenced using 5'-terminal 32p-labelling with gamma-32p ATP and T4 polynucleotide kinase, fingerprinting and controlled nuclease P1 digestion. Modified nucleotides have not been detected in 5'-positions of these oligonucleotides. PSTV consists of about 359 nucleotides and contains a remarkable stretch of 18 purines, mainly adenosines; there is no AUG initiation triplet present. The established oligonucleotide sequences preclude a perfect intramolecular base complementarity within the covalently closed viroid circle. Therefore, the rigid, rod-like native secondary structure of PSTV, as seen in the electron microscope, must be based on a defective rather than on a homogeneous RNA helix. The detailed analysis of the bisulfite-catalized modification of cytidine to uridine in PSTV revealed a higher reactivity for the majority of the cytidines than would be expected for a perfect helix. Since only cytidines in single-stranded regions are knonw to be fully reactive, this finding provides additional evidence for defects in the helical secondary structure of PSTV.
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PMID:Studies on the primary and secondary structure of potato spindle tuber viroid: products of digestion with ribonuclease A and ribonuclease T1, and modification with bisulfite. 41 83

Purified rat liver mitochondria were shown to synthesize poly(adenylic acid) (poly(A)) in vitro. Detection of the poly(A) synthesizing activity was facilitated by addition of NaF to the reaction was shown to be poly(A) by its insensitivity to digestion with pancreatic RNase and RNase T1, its degradation by venom phosphodiesterase and its retention on poly (uridylic acid) 20-23 AMP units and it was covalently attached to the endogenous RNA in the mitochondria. Poly(A) synthesis required ATP and a divalent ion and was maximally active in the pH range of 7-8. The reaction was inhibited by atractyloside, cordycepin triphosphate, Rose Bengal, rifamycin derivative AF/103, sodium pyrophosphate, and N-ethylmaleimide. These studies indicate that the mitochondrial poly(A) polymerase previously described in our laboratory (Jacob, S.T., Rose, K.M., and Morris, H.P. (1974), Biochim. Biophys. Acta 361, 312-320) is involved in the posttranscriptional addition of poly(A) sequence to mitochondrial RNA.
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PMID:Poly(adenylic acid) synthesis in isolated rat liver mitochondria. 99 Feb 63

We have used photoaffinity labelling to examine the chloroplast RNA polymerase components which come into contact with nascent transcripts during the in vitro transcription of plastid DNA. The transcripts were synthesized in the presence of a photoactive analogue (4-thio UTP) and alpha-32P-ATP, using enriched pea chloroplast RNA polymerase preparation and a recombinant plasmid containing the plastid 16S rRNA promoter. Brief irradiation of the transcriptional complex crosslinked the photoactive nascent RNA to proximal proteins. Labelling of the transcriptional complex was dependent on 4-thio UTP and template DNA. Two polypeptides of 51 and 54 kDa were consistently crosslinked to the nascent transcripts; about 60% of the total radioactivity of the crosslinked RNA was associated with these polypeptides. In some experiments, two additional polypeptides of 38 and 75 kDa were also found to be associated with about 13% and 17% of the total crosslinked RNA radioactivity, respectively. The UV-crosslinked transcriptional complexes were stable to either DNase or S1 nuclease hydrolysis but partially sensitive to RNase T1. Insensitivity of the complex to hydrolysis with RNase H suggested that the nascent transcripts were not crosslinked to the template. The complexes could also be hydrolysed by proteinase K and thermolysin. No crosslinkage was observed when labelled RNA molecules containing 4-thio UMP residues were added after synthesis to the polymerase preparation. This suggested that the method identified only those polypeptides which came into close contact with the transcript during its synthesis. Antibodies raised against the RNA-protein complex confirmed the presence of the polypeptides in the chloroplast RNA polymerase preparation on Western blots. Preincubation of these antibodies with the chloroplast RNA polymerase inhibited plastid DNA transcription. These data showed that the transcript-binding polypeptides were functional components of the chloroplast transcriptional complex.
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PMID:Photoaffinity labelling of the pea chloroplast transcriptional complex by nascent RNA in vitro. 171 36

Stable association of U2 snRNP with the branchpoint sequence of mammalian pre-mRNAs requires binding of a non-snRNP protein to the polypyrimidine tract. In order to determine how U2 snRNP contacts this protein, we have used an RNA containing the consensus 5' and the (Py)n-AG 3' splice sites but lacking the branchpoint sequence so as to prevent direct U2 snRNA base pairing to the branchpoint. Different approaches including electrophoretic separation of RNP complexes formed in nuclear extracts, RNase T1 protection immunoprecipitation assays with antibodies against snRNPs and UV cross-linking experiments coupled to immunoprecipitations allowed us to demonstrate that at least three splicing factors contact this RNA at 0 degree C without ATP. As expected, U1 snRNP interacts with the region comprising the 5' splice site. A protein of approximately 65,000 molecular weight recognizes the RNA specifically at the 5' boundary of the polypyrimidine tract. It could be either the U2 auxiliary factor (U2AF) (Zamore and Green (1989) PNAS 86, 9243-9247), the polypyrimidine tract binding protein (pPTB) (Garcia-Blanco et al. (1989) Genes and Dev. 3, 1874-1886) or a mixture of both. U2 snRNP also contacts the RNA in a way depending on p65 binding, thereby further arguing that the latter may correspond to the previously characterized U2AF and pPTB. Cleavage of U2 snRNA sequence by a complementary oligonucleotide and RNase H led us to conclude that the 5' terminus of U2 snRNA is required to ensure the contact between U2 snRNP and p65 bound to the RNA. More importantly, this conclusion can be extended to authentic pre-mRNAs. When we have used a human beta-globin pre-mRNA instead of the above artificial substrate, RNA bound p65 became precipitable by anti-(U2) RNP and anti-Sm antibodies except when the 5' end of U2 snRNA was selectively cleaved.
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PMID:The 5' end domain of U2 snRNA is required to establish the interaction of U2 snRNP with U2 auxiliary factor(s) during mammalian spliceosome assembly. 185 Jan 27

The interferon-induced enzyme 2-5A synthetase is shown to adenylate tRNA. Yeast tRNAPhe was incubated with the enzyme in the presence of double stranded RNA (in this case polyI-polyC) and ATP or deoxyATP. The reaction products were analyzed by ribonuclease T1 digestion of the tRNA, polyacrylamide gel electrophoresis and autoradiography. Using ATP, the 2-5A synthetase adds one, two or three AMP residues to the 3'-end of the tRNA whereas when dATP is replacing ATP, only one nucleotide unit is added. It is concluded that one of the mechanisms of the interferon-induced antiviral effect may be an inhibition of the translation process caused by an inactivation of tRNA molecules by a 2-5A synthetase catalyzed 2'-adenylation of the 3'-end.
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PMID:The interferon-induced enzyme 2-5A synthetase adenylates tRNA. 241 7

As demonstrated by RNase T1 protection assays at 0 degrees C without ATP, U1 and U5 snRNPs purified by isopycnic centrifugation in cesium chloride bind to the 5' and 3' splice sites of human beta-globin pre-mRNA, respectively. We also devised a saturation-complementation assay and have found that this purified U5 snRNP, unlike U1, successfully competes with snRNP-free fractions of nuclear proteins which inhibit spliceosome assembly and splicing. Restoration of activity requires intact U5 snRNA and correlates with the presence of the 100 Kd intron binding protein (IBP) which we have previously characterized (Tazi et al., 1986, Cell 47, 755-766). Our results are compatible with a model in which the recognition of the 3' splice site by IBP-U5 snRNP is one of the earliest events of the spliceosome assembly. It could organize the structure of the 3' splice site region of the human beta-globin like pre-mRNAs. However, on the basis of results showing that beta-globin and major late adenovirus seem to have different requirements with respect to IBP-U5 snRNP, it appears that some pre-mRNAs could have a native structure that necessitates less if at all IBP-U5.
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PMID:Purified U5 small nuclear ribonucleoprotein can relieve the inhibition of spliceosome assembly and splicing by snRNP-free nuclear proteins. 252 52

The binding of hnRNP proteins to pre-mRNAs in nuclear extracts, and as isolated proteins, was studied by using monoclonal antibody immunopurification of hnRNP proteins bound to RNase T1-generated fragments. Several major hnRNP proteins, A1, C and D, bind specifically to the 3' end of introns within a region containing the conserved polypyrimidine stretch between the branch site and the 3' splice site. Mutations which alter the conserved 3' splice site dinucleotide AG strongly impair or abolish the binding of the A1 protein as well as of an anti-Sm reactive component(s) to this region. The A1, C and D proteins do not bind efficiently to fragments of either bacterial RNA or the intronless spliced product (mRNA). The binding of these proteins at the 3' end of the intron does not require addition to the extract of exogenous ATP, but remains after ATP addition. These findings demonstrate that several hnRNP proteins have RNA binding specificities on pre-mRNA, and suggest a model for hnRNP particle structure and assembly.
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PMID:RNA binding specificity of hnRNP proteins: a subset bind to the 3' end of introns. 320 40


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