Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Incubation of CMP in 2H2O with 0.5M cysteine methyl ester at p2H 5 and 37 degrees C for 24 h resulted in 43% exchange of 5-H to 5-2H. No deamination of the cytosine nucleus was noted during this treatment. Native and denatured DNA samples from calf thymus were treated in 3H2O with cysteine methyl ester at pH 5 and 37 degrees C for 24 h and incorporation of tritium into each DNA base was determined by enzymic digestion of the treated DNA. The order of the specific radioactivity found was cytosine greater than guanine greater than adenine greater than thymine for denatured DNA and guanine greater than adenine approximately cytosine greater than thymine for native DNA. The ratio of radioactivity for denatured/native was 11.6 for cytosine, 1.5 for guanine, 1.8 for adenine and 1.1 for thymine. Hence the incorporation in cytosine under the reaction conditions is preferential for single-stranded, nonhelical regions of DNA. Escherichia coli glutamic acid tRNA II was treated in 3H2O with 1.24 M cysteine methyl ester at pH 5 and 37 degrees C. The 24-h-treated tRNA was digested with ribonuclease T1 and the fragments were fractionated. Each fragment was then digested with ribonuclease T2 into mononucleotides and the radioactivity distribution among the bases was determined. The average radioactivity found for each of the bases of the four major nucleotides was cytosine greater than guanine approximately adenine greater than uracil. The radioactivity in cytosine varied greatly among the RNase T1 fragments, the ratio of the highest to the lowest radioactivity being 18.7. The corresponding value for guanine was 11.1, for adenine 4.73 and for uracil 3.64. Based on the data obtained, it was deduced that in this tRNA the anticodon loop, the dihydrouridine loop and the extra loop were "exposed" under the conditions employed for the labeling. The 5'-terminal cytosine of the anticodon loop was in a "non-exposed" state, a situation similar to that previously reported for E. coli tyrosine tRNA [Cashmore, A. R., Brown, D. M. & Smith, J. D. (1971) J. Mol. Biol. 59, 359-373] and for E. coli formylmethionine tRNA [Goddard J. P.+Schulman L. H. (1972) J. Biol. Chem. 247, 3864-3867]. Both cytosine 48, located at the 3'-terminal of the extra loop, and guanine 15 in the dihydrouridine loop were in an "emposed" state. This finding does not agree with a tRNA model in which this pair of cytosine and guanine, commonly found in tRNA sequences, forms hydrogen bondings. Positions 30--32, 61--64 and 71, which are located in the stems, were found to be strongly "buried".
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PMID:Conformation of Escherichia coli glutamic acid tRNA II as studied by hydrogen-tritium exchange catalyzed by cysteine methyl ester. 0 69

Poliovirus was grown in HeLa cells in the presence of phosphorus-32 and actinomycin D. Three to four hours after infection, viral mRNA was recovered from polyribosomes and its identity verified by two-dimensional gel electrophoresis of RNase T1 digests. Digestion of the viral [32P]mRNA with RNase T2 and separation of the products by ion exchange chromatography at pH 5 yielded pUp as possible 5' terminus but no "capping group" of the structure m7G(5')ppp(5')Np. Total cytoplasmic [32P]RNA of HeLa cells, on the other hand, was found to contain capping groups. Neither the capping group nor ppNp or pppNp was found in an RNase T2 digest of poliovirion [32P]RNA, in agreement with previous results [Wimmer, E. (1972) J. Mol. Biol. 68, 537-540]. The data indicate that 5'-terminal m7G(5')ppp(5')Np is absent from poliovirus RNAs and, therefore, is not involved in poliovirus protein synthesis.
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PMID:The 5' end of poliovirus mRNA is not capped with m7G(5')ppp(5')Np. 17 6

The abilities of purine- and pyrimidine-requiring mutants to produce six orthophosphate repressible extracellular enzymes, alkaline phosphatase, 5'-nucleotidase, acid phosphatase, two nucleases and ribonuclease N1 were examined by culturing these mutants in low and high phosphate media containing nucleotide or nucleoside. All the purine requiring mutants produced significantly reduced amounts of alkaline phosphatase, 5'-nucleotidase, acid phosphatase, alkaline nuclease and acid nuclease ranging 0.5-4.2, 5.0-17.4, 25.0-100, 20.3-67.5 and 6.2-48.5%, respectively. Production of ribonuclease N1 was found to be rather stimulated (150-564%) in these mutants. Essentially the same results were obtained for pyrimidine requiring mutants. Among those mutants ad-2 and ad-9 showed relatively high enzyme producing activity. Especially the production of ribonuclease N1 in ad-2 and ad-9 ranged to 4.9- and 5.6-fold that in the wild type. Though nuc-1 mutant (A1) has no ability to produce all these six repressible enzymes, double mutants A1ad-2 and A1ad-9 produced a significant amount of ribonuclease N1 in low and high phosphate media and acid phosphatase in low phosphate media.
Mol Gen Genet 1977 Feb 28
PMID:Control of the Production of orthophosphate repressible extracellular enzymes in Neurospora crassa. 19 39

Nucleotide sequences around kethoxal-reactive guanine residues of 23S RNA in 50S ribosomal subunits have been determined. By use of the diagonal paper electrophoresis method )Noller, H.F. (1974), Biochemistry 13, 4694-4703), 41 ribonuclease T1 oligonucleotides, originating from about 25 sites, were identified and sequenced. These sites are single stranded and accessible in free 50S subunits, and are thus potential sites for interaction with functional ligands during protein synthesis. Examination of these sequences for potential intermolecular base-pairing reveals the following: (1) There are 19 possible complementary combinations between exposed sequences in 16S and 23S RNA containing more than 4 base pairs: 15 containing 5 base pairs and 4 containing 6 base pairs. Nine of these complementary combinations contain 16S RNA sequences which we have previously shown to be protected from kethoxall by 50S subunits (Chapman, N.M., and Noller, H.F. (1977), J. Mol. Biol. 109, 131-149). (2) One of the exposed sites in 23S RNA has a sequence which is complementary to the invariant GT psi CR sequence in tRNA.
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PMID:Nucleotide sequences of accessible regions of 23S RNA in 50S ribosomal subunits. 33 46

Terminal labeling of embryonic feather keratin mRNA with [3H]KBH4 followed by digestion with ribonuclease T1 and T2, alkaline phosphatase, snake venom phosphodiesterase, and nucleotide pyrophosphatase and analysis of the products by high voltage paper electrophoresis, indicated the presence of the sequence m7G(5')ppp(5')N at the 5'-end of the mRNA. Ribonuclease T1 and A digests of the terminally labeled, and also of unlabeled mRNA followed by fractionation on denaturing polyacrylamide gels indicated the presence of polyadenylate tracts ranging in size from 45 to 165 nucleotide at the 3'-end of the mRNA.
Mol Biol Rep 1979 Aug 31
PMID:The terminal structures of feather keratin mRNA. 49 58

Transcription of Escherichia coli ribosomal DNA introduced into Proteus mirabilis on F14 is described. We have developed an assay for E. coli coded ribosomal RNA involving fingerprinting of ribonuclease T1 digests of RNA isolated from ribosomal subunits. Sequence differences in the ribosomal RNA of the two species have allowed us to detect E. coli coded 16S, 23S, and 5S ribosomal RNA in ribosomal subunits of the E. coli-P. mirabilis hybrid. The proportion of E. coli coded rRNA in the hybrid is found at a level which is compatible with the number of E. coli (and P. mirabilis) ribosomal DNA sequences. The resulting ribosomal RNA appears in ribosomes in a form which indicates extensive compatibility of E. coli coded ribosomal RNA with P. mirabilis ribosomal proteins and maturational factors.
Mol Gen Genet 1976 Aug 19
PMID:Transcription of Escherichia coli ribosomal DNA in Proteus mirabilis. 78 56

A procedure for the isolation and purification of a specific hybrid between rat 28S and 18S ribosomal RNA's and nucleolar DNA is described. The method employed includes the following steps: 1) isolation of the nucleolar DNA, 2) hybridization of [14C]rRNA with the nucleolar DNA, and 3) isolation and purification of the rRNA-DNA hybrid complex by chromatography on hydroxylapatite and centrifugation in a CsCl density gradient. In the isolated hybrid complex the RNA:DNA ratio is close to 1:1, and the degree of enrichment of the DNA by the rRNA cistrons is about 1500 times. The hybrid obtained has a sedimentation constant on the order of 20S, is resistant to the action of pancreatic RNase and RNase T1 and sheep brain DNase, and is characterized by high thermostability. Acording to the physicochemical tests used, the rRNA-DNA hybrid complex is a double-stranded poly-nucleotide with an ordered secondary structure.
Mol Biol (Mosk)
PMID:Isolation of a hybrid between rat ribosomal RNA and DNA. 102 44

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a non-cognizable ribonucleotide, 2'AMP, has been determined and refined to an R-factor of 0.159 using X-ray diffraction data at 1.7 A resolution. A specific complex of the enzyme with 2'GMP was also determined and refined to an R-factor of 0.173 at 1.9 A resolution. The adenine base of 2'AMP was found at a base-binding site that is far apart from the guanine recognition site, where the guanine base of 2'GMP binds. The binding of the adenine base is mediated by a single hydrogen bond and stacking interaction of the base with the imidazole ring of His92. The mode of stacking of the adenine base with His92 is similar to the stacking of the guanine base observed in complexes of ribonuclease T1 with guanylyl-2',5'-guanosine, reported by Koepke et al., and two guanosine bases, reported by Lenz et al., and in the complex of barnase with d(GpC), reported by Baudet & Janin. These observations suggest that the site is non-specific for base binding. The phosphate group of 2'AMP is tightly locked at the catalytic site with seven hydrogen bonds to the enzyme in a similar manner to that of 2'GMP. In addition, two hydrogen bonds are formed between the sugar moiety of 2'AMP and the enzyme. The 2'AMP molecule adopts the anti conformation of the glycosidic bond and C-3'-exo sugar pucker, whereas 2'GMP is in the syn conformation with C-3'-endo-C'-2'-exo pucker. The mutation enhances the binding of 2'GMP with conformational changes of the sugar ring and displacement of the phosphate group towards the interior of the catalytic site from the corresponding position in the wild-type enzyme complex. Comparison of two crystal structures obtained provides a solution to the problem that non-cognizable nucleotides exhibit unexpectedly strong binding to the enzyme, compared with high specificity in nucleolytic activity. The results indicate that the discrimination of the guanine base from the other nucleotide bases at the guanine recognition site is more effective than that estimated from nucleotide-binding experiments so far.
J Mol Biol 1992 Feb 20
PMID:Three-dimensional structure of a mutant ribonuclease T1 (Y45W) complexed with non-cognizable ribonucleotide, 2'AMP, and its comparison with a specific complex with 2'GMP. 131 85

In the genetically mutated ribonuclease T1 His92Ala (RNase T1 His92Ala), deletion of the active site His92 imidazole leads to an inactive enzyme. Attempts to crystallize RNase T1 His92Ala under conditions used for wild-type enzyme failed, and a modified protocol produced two crystal forms, one obtained with polyethylene glycol (PEG), and the other with phosphate as precipitants. Space groups are identical to wild-type RNase T1, P2(1)2(1)2(1), but unit cell dimensions differ significantly, associated with different molecular packings in the crystals; they are a = 31.04 A, b = 62.31 A, c = 43.70 A for PEG-derived crystals and a = 32.76 A, b = 55.13 A, c = 43.29 A for phosphate-derived crystals, compared to a = 48.73 A, b = 46.39 A, c = 41.10 A for uncomplexed wild-type RNase T1. The crystal structures were solved by molecular replacement and refined by stereochemically restrained least-squares methods based on Fo greater than or equal to sigma (Fo) of 3712 reflections in the resolution range 10 to 2.2 A (R = 15.8%) for the PEG-derived crystal and based on Fo greater than or equal to sigma (Fo) of 6258 reflections in the resolution range 10 to 1.8 A (R = 14.8%) for the phosphate-derived crystal. The His92Ala mutation deletes the hydrogen bond His92N epsilon H ... O Asn99 of wild-type RNase T1, thereby inducing structural flexibility and conformational changes in the loop 91 to 101 which is located at the periphery of the globular enzyme. This loop is stabilized in the wild-type protein by two beta-turns of which only one is retained in the crystals obtained with PEG. In the crystals grown with phosphate as precipitant, both beta-turns are deleted and the segment Gly94-Ala95-Ser96-Gly97 is so disordered that it is not seen at all. In addition, the geometry of the guanine binding site in both mutant studies is different from "empty" wild-type RNase T1 but similar to that found in complexes with guanosine derivatives: the Glu46 side-chain carboxylate hydrogen bonds to Tyr42 O eta; water molecules that are present in the guanine binding site of "empty" wild-type RNase T1 are displaced; the Asn43-Asn44 peptide is flipped such that phi/psi-angles of Asn44 are in alpha L-conformation (that is observed in wild-type enzyme when guanine is bound).(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1992 Apr 05
PMID:His92Ala mutation in ribonuclease T1 induces segmental flexibility. An X-ray study. 131 2

On the basis of molecular dynamics and free-energy perturbation approaches, the Glu46Gln (E46Q) mutation in the guanine-specific ribonuclease T1 (RNase T1) was predicted to render the enzyme specific for adenine. The E46Q mutant was genetically engineered and characterized biochemically and crystallographically by investigating the structures of its two complexes with 2'AMP and 2'GMP. The ribonuclease E46Q mutant is nearly inactive towards dinucleoside phosphate substrates but shows 17% residual activity towards RNA. It binds 2'AMP and 2'GMP equally well with dissociation constants of 49 microM and 37 microM, in contrast to the wild-type enzyme, which strongly discriminates between these two nucleotides, yielding dissociation constants of 36 microM and 0.6 microM. These data suggest that the E46Q mutant binds the nucleotides not to the specific recognition site but to the subsite at His92. This was confirmed by the crystal structures, which also showed that the Gln46 amide is hydrogen bonded to the Phe100 N and O atoms, and tightly anchored in this position. This interaction may either have locked the guanine recognition site so that 2'AMP and 2'GMP are unable to insert, or the contribution to guanine recognition of Glu46 is so important that the E46Q mutant is unable to function in recognition of either guanine and adenine.
J Mol Biol 1992 May 20
PMID:RNase T1 mutant Glu46Gln binds the inhibitors 2'GMP and 2'AMP at the 3' subsite. 135 Jun 42


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