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)

The accessibility of nucleotides in Escherichia coli tRNAfMet to chemical and enzymatic probes in the presence and absence of methionyl-tRNA synthetase has been investigated. Dimethyl sulfate was used to probe the reactivity of cytosine and guanosine residues. The N-3 position of the wobble anticodon base, C34, was strongly protected from methylation in the tRNA-synthetase complex. A synthetase-induced conformational change in the anticodon loop was suggested by the enhanced reactivity of C32 in the presence of enzyme. Cytosine residues in the dihydrouridine loop and in the 3'-terminal CCA sequence showed little or no change in reactivity. Methylation of the N-7 position of guanosine residues G42, G52, and G70 was partially inhibited by the synthetase. Nuclease digestion of tRNAfMet with alpha-sarcin in the presence of 1-2 mM Mg2+ resulted in cleavage mainly at C71 in the acceptor stem and was strongly inhibited by synthetase. Other nuclease digestion experiments using the single strand specific nucleases RNase A and RNase T1 revealed weak protection of nucleotides in the D loop and strong protection of nucleotides in the anticodon on complex formation. The present data, together with previous structure-function studies on this system, indicate strong binding of methionyl-tRNA synthetase to the anticodon of tRNAfMet, leading to a change in the conformation of the anticodon loop and stem. We propose that this, in turn, produces more distant, and possibly relatively subtle, conformational changes in other parts of the tRNA structure that ultimately lead to proper orientation of the 3' terminus of the tRNA with respect to the active site of the enzyme.
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PMID:Study of the interaction of Escherichia coli methionyl-tRNA synthetase with tRNAfMet using chemical and enzymatic probes. 309 57

Ribonuclease T1 (RNase T1, EC 3.1.27.3) is a guanosine-specific ribonuclease that cleaves the 3',5'-phosphodiester linkage of single-stranded RNA. It is assumed that the reaction is generated by concerted acid-base catalysis between residues Glu-58 and His-92 or His-40. From the results of chemical modification and NMR studies, it appeared that the residue Glu-58 was indispensable for nucleolytic activity. However, we have recently demonstrated that Glu-58 is an important but not an essential residue for catalytic activity, using the methods of genetic engineering to change Glu-58 to Gln-58 etc [Nishikawa, S., Morioka, H., Fuchimura, K., Tanaka, T., Uesugi, S., Ohtsuka, E., & Ikehara, M. (1986) Biochem. Biophys. Res. Commun. 138, 789-794]. In the present paper, we report that mutants of RNase T1 with residue Ala-40 or Ala-92 have almost no activity, while mutants that contain Ala-58 retain considerable activity. These results show that the two histidine residues, His-40 and His-92, but not Glu-58, are indispensable for the catalytic activity of the enzyme. We propose a revised reaction mechanism in which two histidine residues play a major role, as they do in the case of RNase A.
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PMID:Two histidine residues are essential for ribonuclease T1 activity as is the case for ribonuclease A. 312 7

Procedures are described for identification of very infrequent in vivo 3'-ends of RNA. After purification by filter hybridization, the 3'-ends were labeled with [5'-32P] cytosine-3'-P in the RNA ligase reaction. Significantly fewer counts were incorporated in the ligase reaction than in the polynucleotide kinase reaction to label 5'-ends. The incorporation was increased by increasing the RNA concentration 5-10 fold by using only one round of filter hybridization. Non-specific RNA binding could be eliminated by RNase A treatment of the filter if a great excess of denatured heterologous DNA was immobilized along with the DNA probe. Significant amounts of DNA were released when eluting the hybrid RNA from such filters. DNA inhibited the ligase reaction, while its DNase products were even more inhibitory. Treatment of the DNase products with alkaline phosphatase completely eliminated the inhibition. We detected no spurious 5'- or 3'-ends generated in the hybrid RNA by RNase A activity used to reduce the non-specific RNA. Also, RNase T1 could be used in place of RNase A to eliminate non-specific RNA binding, but about 25 times more RNase T1 (microgram/microgram) was needed. We used partial alkali digestion to sequence 3'-ends. A major (one hit) and minor (two hit) set of products were produced which could be distinguished from each other by alkaline phosphatase treatment and homochromatography of the products.
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PMID:Isolating and sequencing the infrequent 3'-ends of a specific mRNA. 331 56

Dexamethasone-receptor complexes from HeLa cell cytosol sediment at 7.4S in low salt sucrose gradients, and at 3.8S in high salt gradients. If cytosol is heated at 25 degrees C, receptor complexes sediment at 6.9S in low salt, and at 3.6S in high salt gradients. RNase A treatment at 25 degrees C, instead, results in receptor complexes which sediment in low salt gradients as two major forms at 6.5 and 4.8S. Receptor complexes from RNase A-treated cytosols sediment as their counterparts from untreated cytosols in high salt gradients. Although the shift in sedimentation properties of receptor complexes at 2 degrees C is induced by RNase A, and not by other low molecular weight basic proteins or RNase T1, the effect can be also obtained by inactive RNase A. The catalytically active enzyme, however, is required to observe 6.5 and 4.8S complexes after cytosol incubations at 25 degrees C. Placental ribonuclease inhibitor prevents the appearance of RNase A-induced receptor forms at 25 degrees C, but not at 2 degrees C. Moreover, this inhibitor can prevent the 7.4 to 6.9S shift in sedimentation coefficient of receptor complexes caused by cytosol heating. Dexamethasone-receptor complexes from HeLa cell cytosol show low levels of binding to DNA-cellulose, and heating at 25 degrees C is required to observe a six-fold increase in DNA binding levels. RNase A treatment of cytosols at 2 degrees C does not result in significant enhancement in receptor complex binding to DNA. If RNase A treatment is carried out at 25 degrees C, however, DNA binding levels of receptor complexes increased by 25% over the values observed with control heated cytosol. This effect cannot be observed if RNase T1 substitutes for RNase A. Placental ribonuclease inhibitor can prevent the temperature-dependent increase in DNA binding properties of dexamethasone-receptor complexes either in the presence or absence of exogenous RNase A. These findings indicate that exogenous RNases can perturb the structure of dexamethasone-receptor complexes without being involved in the transformation process.
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PMID:RNase A effects on sedimentation and DNA binding properties of dexamethasone-receptor complexes from HeLa cell cytosol. 391 16

Poliovirus RNA that has been derivatized at the 3'-end with NaIO(4)-NaB(3)H(4) yields, after hydrolysis with alkali or RNase T2, predominantly labeled residues of modified adenosine; no labeled nucleoside derivative is produced by digestion with RNase A or RNase T1. The 3'-terminal bases of the RNA are, therefore,...ApA(OH). Hydrolyzates of poliovirus [(32)P]RNA, after exhaustive digestion with RNase T1 or RNase A, contain, besides internal oligonucleotides, polynucleotides resistant to further action of ribonucleases T1 and A, respectively; these polynucleotides were isolated by membrane-filter binding or ion-exchange chromatography. The sequence of the T1-resistant polynucleotide was determined to be (Ap)(n)A(OH), that of the RNase A-resistant polynucleotide was GpGp(Ap)(n)A(OH). The chain length (n) of the polyadenylic acid, as analyzed by different methods, averages 89 nucleotides. Gel electrophoresis revealed heterogeneity of the size of poly(A). Poliovirus RNA, when labeled in vitro at the 3'-end, contains [3'-(3)H]poly(A); when labeled in vivo with [(3)H]A, it contains [(3)H](Ap)(n)A(OH). The data establish that... YpGpGp(Ap)([unk])A(OH) is the 3'-terminal sequence of poliovirus RNA, Type 1 (Mahoney). Since this mammalian virus reproduces in the cell cytoplasm, these observations may modify prior interpretations of the function of polyadenylate ends on messenger RNAs.
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PMID:Polyadenylic acid at the 3'-terminus of poliovirus RNA. 434 Jan 62

Adenosine is the major 3'OH-terminal nucleoside of the 60-70S RNA genome of the murine sarcoma-leukemia virus, its 30-40S RNA subunits, and the poly(A) segments derived by RNase treatment of both RNA species, as determined by periodate oxidation-[(3)H]-borohydride reduction. The binding 30-40S RNA to oligo(dT)-cellulose suggests that most viral RNA subunits contain poly(A). The molecular weight of poly(A) derived from viral RNA by digestion with RNase and purified by affinity chromatography is 64,000-68,000, as determined by gel electrophoresis. From the size of poly(A) and the poly(A) content of viral RNA (1.6%), it is estimated that there is about one poly(A) segment for each viral 30-40S RNA subunit. The results of 3'-termini labeling with [(3)H]borohydride, in vivo labeling with [(3)H]adenosine, and base composition of [(32)P]poly(A) indicate that a homopoly(A) segment is located at the 3'-end of a 30-40S RNA subunit. The homogeneous poly(A) segments isolated from RNase T1 digests of 60-70S [(32)P]RNA consist of one cytidylate, one uridylate, and about 190 adenylate residues, while those isolated from RNase A digests consist exclusively of adenylate residues. These results indicate that -G(C,U)A(190)A(OH) is the 3'-terminal nucleotide sequence of the viral 30-40S RNA subunits.
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PMID:The homopolyadenylate and adjacent nucleotides at the 3'-terminus of 30-40s RNA subunits in the genome of murine sarcoma-leukemia virus. 436 65

The 5'-terminal structures of human adenovirus type 2 (Ad2) early region 2 (E2) mRNA were investigated. The E2 transcription unit has several interesting properties, including the presence of a TATA-like box that matches the consensus sequence poorly, delayed transcription during early stages of infection, and a switch in promoter recognition late after infection. E2-specific RNA, 5'-labeled in vitro to high specific activity was analyzed. Purified E2 mRNA was digested with RNase A or RNase T1 and the resulting oligonucleotides were resolved by two dimensional paper electrophoresis-homochromatography. Remarkably, as many as sixteen 5'-terminal RNase A oligonucleotides were identified and their sequences were deduced. The most common 5'-termini in the RNase A digest were p(m6)AmCp, p(m6)AmA(m)Cp, pGmA(m)Cp, and p(m6)AmG(m)Cp. Two RNase A oligonucleotides originated from the E4 promoter region, consistent with electron microscopic observations. The sequence encoding these potential initiation sites covered about 90 nucleotides. Eleven of the sequences of the 5'-terminal RNase A oligonucleotides were aligned with the Ad2 DNA sequence in the Ad2 E2 promoter region. If the heterogeneous termini in the E2 promoter region were generated by a process of transcription initiation, their existence cannot be explained by stuttering of RNA polymerase II. This suggests that the transcription of Ad early region 2 has features which differ from those of other Ad2 early gene transcription units. Perhaps this is due to the absence of a conventional TATA box which is believed to position the initiation site. Alternatively, it is conceivable that the E2 promoter represents an alternate class of RNA polymerase II promoters containing different signals with different requirements for activation and/or that an E1A gene product modifies transcription initiation.
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PMID:Unusual heterogeneity of the 5'-termini of human adenovirus type 2 early region E2 mRNA. 608 49

The dideoxynucleotide method for sequencing DNA developed by Sanger et al. [Sanger, F., Nicklen, S. & Coulson, A. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467] was modified to allow sequence analysis of poliovirus RNA without recourse to cloning. Our method involves reverse transcription of poliovirus RNA followed by cDNA-dependent DNA synthesis in the presence of unlabeled dNTPs and 2',3'-dideoxynucleoside triphosphates, with Escherichia coli DNA polymerase I (Klenow) used to catalyze the reaction. DNA synthesis is primed by 5'-32P-labeled RNase T1- or RNase A-resistant oligonucleotides generated from poliovirus RNA. The sequence of 1060 nucleotides preceding the 3'-terminal poly(A) is presented. Based on the position of termination codons we propose that viral translation terminates at nucleotide -562.
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PMID:Sequence of 1060 3'-terminal nucleotides of poliovirus RNA as determined by a modification of the dideoxynucleotide method. 615 42

A lysed cell system was used to study the organelle structure and nucleation of exogenous tubulin at kinetochores and centrosomes in mitotic PtK2 cells. We have used this lysed cell system in conjunction with nuclease digestion experiments to determine which specific nucleic acids (DNA or RNA) are involved in either the structure and/or microtubule-initiating capacity of kinetochores and centrosomes. The results indicate that DNase I specifically decondenses the kinetochore plate structure, with the eventual loss in the ability of the chromosomes to nucleate microtubule assembly. DNase I had no effect on either the structure or nucleating capacity of centrosomes. Both RNase T1 and RNase A specifically attacked the amorphous pericentriolar material of the centrosomes, with a concomitant loss in the ability of this material to nucleate microtubule formation. Neither RNase appeared to affect the structure or nucleating capacity of the kinetochore. Therefore, the two types of nucleases appear to exert preferential effects on the different types of microtubule initiation sites in mitotic mammalian cells. The results suggest that DNA is a major component of the kinetochore, while RNA is a major component of the amorphous pericentriolar material. These findings support the concept that microtubule initiation sites in mitotic cells contain nucleic acids which are essential for the structural and functional integrity of the sites.
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PMID:Tubulin nucleation and assembly in mitotic cells: evidence for nucleic acids in kinetochores and centrosomes. 618 68

In vivo transcription and polyadenylation at the junction of the L cistron and the 5'-terminal extracistronic region of vesicular stomatitis virus RNA was investigated. Annealing of 5'32P-labeled RNA representing the 5'-terminal noncoding 77 nucleotides of vesicular stomatitis virus genomic RNA to L gene mRNA resulted in specific duplex formation. Two specific RNase T1- and RNase A resistant duplexes, 66 and 77 nucleotides long, bound to oligodeoxythymidylic acid cellulose. The specific sizes of the duplexes and their selection by oligodeoxythymidylic acid cellulose chromatography demonstrated that they were covalently linked to the polyadenylic acid tail of L gene mRNA. These data strongly suggest that the viral polymerase polyadenylates L gene mRNA in vivo by using the stretch of seven uridine residues at the end of the L cistron and that the polymerase can resume transcribing the 5'-terminal extracistronic region, resulting in a covalent linkage of the transcript to the polyadenylic acid tail of L gene mRNA.
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PMID:In vivo transcription of the 5'-terminal extracistronic region of vesicular stomatitis virus RNA. 626 2


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