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)

Globin messenger RNA, isolated from human peripheral blood reticulocytes, was transcribed into complementary DNA by use of the RNA-dependent DNA polymerase of avian myeloblastosis virus. The complementary DNA was then transcribed into (32)P-labeled complementary RNA by E. coli RNA polymerase in the presence of alpha-(32)P-labeled ribonucleoside triphosphates. The fingerprint pattern obtained from ribonuclease T1 digests of human globin complementary RNA was specific and reproducible. Different patterns were obtained from digests of duck, mouse, and rabbit globin complementary RNA. The fingerprint patterns obtained from digests of purified natural human 10S globin messenger RNA, labeled in vitro with (125)I or with [gamma-(32)P]ATP and polynucleotide kinase, were similar to that of the complementary RNA but contained some additional oligonucleotides. Sufficient nucleotide sequence information has been obtained from about 50% of the intermediate sized oligonucleotides (8-14 base residues long), to make possible examination of correspondence between these nucleotide sequences and globin amino-acid sequences. Approximately 70% of these oligonucleotide sequences can be matched to unique amino-acid sequences in the alpha- or beta-globin chains. The other 30% do not match known amino-acid sequences and presumably correspond to untranslated portions of the mRNA; some of these sequences, however, can be matched to amino-acid sequence in the abnormally long segment of the alpha chain of hemoglobin Constant Spring, which is thought to result from a chain-termination mutation.
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PMID:Nucleotide sequences of human globin messenger RNA. 413 9

An eicosanucleotide C--G--C--G--G--G--G--U--G--G--A--G--C--A--G--C--C--U--G--Gp corresponding to the bases 1--20 of the nascent sequence for the Escherichia coli tRNAfMet has been synthesized by the joining of the chemically synthesized oligonucleotides C--G--C--G, G--G--G--U--G--G and A--G--C--A--G--C--C--U--G--Gp using RNA ligase from T4-infected E. coli. The hexanucleotide and decanucleotide were phosphorylated with polynucleotide kinase and [gamma-32P]ATP prior to the joining reactions. The decanucleotide and eicosanucleotide were reconstituted respectively with the 3'-three-quarter molecule obtained by limited digestion with RNase T1 of the natural tRNAfMet from E. coli and the activity of the complex as a methionine acceptor was tested using purified methionyl-tRNA synthetase from E. coli. The amino acid acceptor activity of the reconstituted molecules was 11% and 84% with respect to that of the intact tRNAfMet.
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PMID:Synthesis of 5' fragments of formylmethionine transfer ribonucleic acid and their reconstitution with a natural three-quarter molecule. 615 76

The major 5'-termini of human adenovirus type 2 early gene block 4 mRNA were sequenced. Poly(A+) polyribosomal RNA was isolated from Ad2 early infected cells, the 5'-terminal m7GPPP removed and the 5'-OH of the penultimate 2'-0-methylated nucleotide labeled with [gamma-32P]ATP using polynucleotide kinase. Ad2 E4 mRNA was purified by hybridization to the Ad2 EcoRI-C fragment and was digested with RNase T1. The resulting oligonucleotides were resolved by two dimensional paper electrophoresis-homochromatography. Four major and 3-4 minor 5'-terminal sequences were identified and characterized. The sequence of the 5'-terminal structures of the major four termini are: (1) m7GpppUmU(m)UUACACUGp, (2) m7GpppUmU(m)UACACUGp, (3) m7GpppUmU(m)ACACUGp, and (4) m7Gppp(m6)AmC(m)ACUGp. These major 5'-terminal sequences were aligned with nucleotide 325, 326, 327, and 329 from the righthand end of the known Ad2 DNA sequence (1) in the region mapped as the 5'-terminus of E4 mRNA by electron microscopy (2,3) and S1 nuclease-gel (4) mapping. Two potential ribosomal binding sites and an initiator codon were found at 40 to 65 nucleotides and about 80 nucleotides, respectively, from these heterogenous 5'-termini. Ad2 E4 major mRNA species appear to be unique since mRNA molecules initiate at a pyrimidine, perhaps by RNA polymerase stuttering, or they are products of an unusual type of RNA processing.
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PMID:Nucleotide sequences and mapping of novel heterogenous 5'-termini of adenovirus 2 early region 4 mRNA. 616 92

Ultraviolet light induced RNA-protein cross-linking for identification of polypeptides interacting with RNA in intact cells (Wagenmakers et al. 1980), is limited by the intensity of the label in the proteins or in residual nucleotides remaining attached to the proteins after RNase treatment of the RNA-protein complexes. Here we report a method, where th cross-linked RNA-protein complexes are treated with RNase T1 and the T1-oligonucleotides covalently linked to the proteins are labeled in the 5' terminus using gamma-32P-ATP and T4 polynucleotide kinase. The cross-linked proteins can then readily be identified owing to the incorporated 32P label. As examples, proteins associated with polyadenylated mRNA, hnRNA and adenoviral VA RNA were identified. A protein with a molecular weight of approximately 50,000 is found associated with adenovirus-coded VA RNA. This was confirmed by binding assays, in which labeled VAI RNA is incubated with proteins from uninfected and adenovirus infected HeLa cells immobilized on nitrocellulose sheets.
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PMID:Detection of a cellular polypeptide associated with adenovirus-coded VA RNA using in vitro labeling of proteins cross-linked to RNA. 617 41

RNA genomes from standard vesicular stomatitis virus and two defective interfering (DI) particles dI 0.33 (DI-T) and DI 0.52, were purified and digested with RNase T1. The resulting oligonucleotides were labeled at the 5' end with [32P]ATP and separated by two-dimensional electrophoresis in polyacrylamide gels. All of the major oligonucleotides containing 20 or more nucleotides were sequenced. Those oligonucleotides that were thought to be in common by their migration on polyacrylamide gels actually did have identical sequences. Those oligonucleotides thought to be unique to the DI RNAs either differed by only one nucleotide from oligonucleotides of the standard RNA or contained new sequences which were complementary to known sequences at the 5' end. These data indicate that RNAs from DI particles are not simple deletions but contain point mutations and additional complementary sequences.
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PMID:Comparison of ribonucleotide sequences from the genome of vesicular stomatitis virus and two of its defective interfering particles. 626 Sep 89

RNA polymerase is known to undergo a cycling process during initiation of transcription in vitro in which large amounts of small oligonucleotides are released. We have used this cycling reaction to determine the 5' end of the RNA synthesized from the outer ends of the Tn5 inverted repeats. By analyzing the size of the radiolabeled oligonucleotides synthesized using different labeled nucleoside triphosphates and in reactions deficient for a particular nucleoside triphosphate, the partial 5' sequence was obtained. This sequence was correlated with the DNA sequence of the region and an unambiguous origin for the mRNA was determined. The start site for the RNA, which is located at 97 base pairs from the outer ends of the inverted repeats, was confirmed by digesting gamma-32 P-ATP labeled full length (run off) transcripts with ribonuclease T1. The resulting gamma-labeled T1 generated oligonucleotide corresponded to the predicted size determined using the cycling reaction. With the knowledge of the RNA start site, the probable translation start sites for the protein(s) known to be required for Tn5 transposition can be predicted. Possible implications of the DNA sequence with respect to the regulation of the transposase are also discussed.
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PMID:Localization of the Tn5 transposase promoter using the cycling reaction of RNA polymerase. 626 98

The 6 S leader RNA transcript from the Escherichia coli threonine operon controlling region was synthesized in vitro using purified RNA polymerase and restriction fragment DNA templates. The terminated leader transcript was analyzed by RNase T1 digestion followed by electrophoresis on 20% polyacrylamide, 8 M urea gels. Oligonucleotides of 7, 8, 13, 15, and 35 bases in length were detected and correlated with the known DNA sequence. The kinetics of RNase T1 digestion indicated that the RNA forms extensive secondary structure, especially at the 3'-terminus of the transcript. The sites of transcription initiation were determined by labeling the 5'-end of the transcript with [gamma-32P]ATP or -GTP followed by direct RNA sequencing. The DNA sequence preceding the initiation site shows homology with the equivalent regions of other bacterial and bacteriophage promoters. The transcription termination sites were determined by mapping of the RNase T1 oligonucleotides arising from the 3'-terminus of the transcript. Comparison of the mobilities of the 3'-oligonucleotides with the mobilities of standards on 20% polyacrylamide, 8 M urea gels indicated that the RNA contains a heterogeneous 3'-terminus. The two predominant oligonucleotides were CU7 and CU8. The 3'-terminus of the transcript also contains a region of dyad symmetry immediately preceding a stretch of uridine residues, characteristic of other rho-independent transcripts. In addition, kinetic studies indicated that RNA polymerase pauses approximately 50 base pairs upstream from the site of termination. The pause site appears to be immediately distal to another region of dyad symmetry.
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PMID:Initiation, pausing, and termination of transcription in the threonine operon regulatory region of Escherichia coli. 627 52

Phage lambda RNA was synthesized in vitro in the presence of [gamma-32P]ATP or GTP. The 12 S, 9 S, 8 S, 6 S, and 5 S transcripts were labeled specifically at the 5'-end with [gamma-32P]ATP, while [gamma-32P]GTP was incorporated at the 5'-end of the 4 S RNA. Transcription of lambda DNA at various salt concentrations was studied by three experimental methods: electrophoresis of the transcripts in polyacrylamide gels; hybridization of RNA to DNA restriction fragments transferred to nitrocellulose paper; and digestion of the transcripts with RNase T1 and analysis of the resulting 5'-end 32P-labeled oligonucleotides by 20% polyacrylamide gel electrophoresis. Transcription from promoters pL, pR, and p'R was maximal between 50 mM and 150 mM NaCl and diminished as the salt concentration was raised to 200 mM. RNA synthesis originating from p'R was predominant at all salt concentrations. The relative salt sensitivity of RNA synthesis was determined for these promoters and found to decrease in the order: pL > pR > p'R.
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PMID:Analysis of the in vitro synthesis of 5'-gamma-32P-labeled transcripts from coliphage lambda by gel electrophoresis, RNA-DNA hybridization, and RNase T1 digestion. 644 58

Bacteriophage lambda transcripts were synthesized in vitro using lambda b2 DNA and Escherichia coli RNA polymerase. RNA molecules initiating with ATP were labeled exclusively at the 5'-end by transcribing in the presence of [gamma-32P]ATP. They were analyzed by electrophoresis in 3.5% polyacrylamide, 7.5 M urea gels followed by autoradiography. The previously known 6 S rho-independent transcript and rho-dependent 9 S and 8 S transcripts were observed. A new rho-dependent 5 S transcript was also detected. The 5 S RNA was not the result of cleavage of larger RNAs by ribonuclease III. Analysis of partial ribonuclease T1 digestion products of isolated 5'-end-labeled transcripts showed that the 5 S RNA is synthesized from promoter pR, the promoter for the 9 S and 8 S RNA species. A similar analysis of transcripts labeled with 32P exclusively at the 3'-terminus by the post-transcriptional addition of a [32P]pCp moiety with T4 RNA ligase revealed the positions of guanosine nucleotides proximal to the 3'-hydroxyl end. This information, and inspection of the known DNA nucleotide sequence downstream from pr, allowed us to conclude that the 5 S RNA is a mixture of molecules 112 and 113 nucleotides long terminating in the middle of gene cro. The nucleotide sequence at the 3'-end of the 5 S RNA is similar to that of other rho-dependent transcripts and lacks the oligo(U) found at the terminus of rho-independent transcripts.
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PMID:Characterization of a rho-dependent termination site within the cro gene of bacteriophage lambda. 644 59

Mammalian RNA polymerase II was shown to utilize dinucleoside monophosphates for priming of promoter specific RNAs. In a reconstituted system containing purified polymerase and HeLa cell fractions, dinucleotides were incorporated by complementarity with template sequences at the in vivo cap sites of the adenovirus major late and adenovirus early region IV promoters. Incorporation was shown by label transfer experiments and by determining the size of 5'-terminal RNase T1-resistant oligonucleotides. All 16 dinucleotides were tested for priming of RNA chains at the major late promoter. RNA polymerase II initiated with various primers over a contiguous region of 9 bases, centered around the in vivo initiation site. We suggest that the polymerase drifts or oscillates over this region. Using a dinucleotide challenge protocol, the rate of initiation at the major late promoter was measured following preincubation of the template DNA with RNA polymerase II and factors. Initiation with ATP was 90% complete within the 1st min after addition of nucleotide triphosphates. Stimulation of transcription by dinucleotides was not observed, due to this rapid initiation. The 5'-hydroxyl terminus of dinucleotide-primed RNAs remained unmodified. Although transcripts initiated with ATP were rapidly capped in whole cell extracts, ATP-primed RNA synthesized in the reconstituted system retained free 5'-terminal phosphates. Thus, capping was not essential for synthesis of long runoff RNAs.
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PMID:Dinucleotide priming of transcription mediated by RNA polymerase II. 658 1


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