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 recombinant Tyr45Trp mutant of Lys25-ribonuclease T1 was overexpressed and purified from an Escherichia coli strain. The mutant enzyme, which shows reduced activity towards GpA and increased activity towards pGpC, pApC and pUpC compared with wild-type RNase T1, was co-crystallized with 2'-adenylic acid by microdialysis. The space group is P212121 with unit cell dimenions a = 4.932(2), b = 4.661(2), c = 4.092(1) nm. The crystal structure was solved using the coordinates of the isomorphous complex of wild-type RNase T1 with 2'-AMP. The refinement was based on Fhkl of 7726 reflexions with Fo greater than or equal to 1 sigma (Fo) in the resolution range of 2.0-0.19 nm and converged with an R factor of 0.179. The adenosine of 2'-AMP is not bound to the guanosine binding site, as could be expected from the mutation of Tyr45Trp, but is stacked on the Gly74 carbonyl group and the His92 imidazole group which form a subsite for substrate binding, as already observed in the wild-type 2'-AMP complex. The point mutation of Tyr45Trp does not perturb the backbone conformation and the Trp-indole side chain is in a comparable position to the phenolic Tyr45 of the wild-type enzyme.
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PMID:Crystal structure of the Tyr45Trp mutant of ribonuclease T1 in a complex with 2'-adenylic acid. 191 64

We have succeeded in crystallizing complexes of a mutant ribonuclease T1 (Y45W) with the non-cognizable ribonucleotides 2'AMP and 2'UMP by macroscopic seeding of microcrystals of the mutant enzyme complexed with 2'GMP, which is the cognizable nucleotide inhibitor. The mutant enzyme has a tryptophan residue instead of Tyr45 of the wild-type enzyme and thus this mutation enhances the binding of ribonucleotides to the enzyme. The space group is P212121 with unit cell dimensions a = 49.40 A, b = 46.71 A, c = 41.02 A for the complex with 2'AMP and a = 48.97, b = 46.58 A, c = 40.97 A for the complex with 2'UMP, both of which are poorly isomorphous to the mother crystals. Diffraction data for the complexes with 2'AMP and 2'UMP were collected on a diffractometer at 1.7 A and 2.4 A resolution, respectively. The present studies show that crystallization of non-specific complexes of other protein-ligand systems with the dissociation constants around 10(-3) M, or even larger, could be feasible by application of the seeding technique. A comparison of the crystal structures of the complexes with that with 2'GMP may serve as a structural basis for the determination of differences between the specific and non-specific interactions of the enzyme.
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PMID:Crystallization and preliminary X-ray investigation of non-specific complexes of a mutant ribonuclease T1 (Y45W) with 2'AMP and 2'UMP. 212 72

We present a calculation of the relative changes in binding free energy between the complex of ribonuclease T1 (RNase Tr) with its inhibitor 2'-guanosine monophosphate (2'GMP) and that of RNase T1-2'-adenosine monophosphate (2'AMP) by means of a thermodynamic perturbation method implemented with molecular dynamics. Using the available crystal structure of the RNase T1-2'GMP complex, the structure of the RNase T1-2'AMP complex was obtained as a final structure of the perturbation calculation. The calculated difference in the free energy of binding (delta delta Gbind) was 2.76 kcal/mol. This compares well with the experimental value of 3.07 kcal/mol. The encouraging agreement in delta delta Gbind suggests that the interactions of inhibitors with the enzyme are reasonably represented. Energy component analyses of the two complexes reveal that the active site of RNase T1 electrostatically stabilizes the binding of 2'GMP more than that of 2'AMP by 44 kcal/mol, while the van der Waals' interactions are similar in the two complexes. The analyses suggest that the mutation from Glu46 to Gln may lead to a preference of RNase T1 for adenine in contrast to the guanine preference of the wild-type enzyme. Although the molecular dynamics equilibration moves the atoms of the RNase T1-2'GMP system about 0.9 A from their X-ray positions and the mutation of the G to A in the active site increases the deviation from the X-ray structure, the mutation of the A back to G reduces the deviation. This and the agreement found for delta delta Gbind suggest that the molecular dynamics/free energy perturbation method will be useful for both energetic and structural analysis of protein-ligand interactions.
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PMID:Calculation of the relative binding free energy of 2'GMP and 2'AMP to ribonuclease T1 using molecular dynamics/free energy perturbation approaches. 215 20

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

A nuclease S1 mapping procedure was used to identify sites accessible to nucleases in the 3'-noncoding region of the rabbit globin mRNAs. A complex structure was evident in the alpha-globin species, with one highly accessible single-stranded site, large portions in an accessible double-stranded configuration, and a portion not accessible to any of the nucleases. In the beta-globin mRNA, the region was more uniformly accessible to RNase T1 and to a cobra venom enzyme specific for double-stranded RNA, but it had only a single site highly accessible to a bulkier Neurospora endonuclease. The patterns of cleavage were nearly identical in the deproteinized mRNAs and in the mRNAs associated with polyribosomes in reticulocyte extracts. In both species, a zone of secondary structure occurred around the poly(A) junction. In each species, virtually all the molecules had a poly(A) sequence of at least 20-25 AMP residues. A periodicity in poly(A) size distribution was observed. These results indicate that the beginning of this sequence is well protected against degradation inside the cell and that zones of partial protection occur at measured intervals. In crude extracts, where the poly(A) is covered with proteins, this sequence was protected against nuclease digestion.
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PMID:Structural features in the 3'-terminal region of polyribosome-bound rabbit globin messenger RNAs. 300 Oct 55

We analyzed the genetic structure and gene products of the newly isolated avian sarcoma virus UR1, which recently has been shown to be replication defective and to contain no sequences homologous to the src gene of Rous sarcoma virus. The sizes of the genomic RNAs of UR1 and its associated helper virus, UR1AV, were determined to be 29S and 35S (5.9 and 8.5 kilobases), respectively, by gel electrophoresis and sucrose gradient sedimentation. RNase T1 oligonucleotide mapping of purified viral RNAs indicated that UR1 RNA contains eight unique oligonucleotides in the middle of the genome and shares four 5'-terminal and three 3'-terminal oligonucleotides with UR1AV RNA. The unique sequences of UR1 and Fujinami sarcoma virus were found to be closely related to each other by molecular hybridization of UR1 RNA with DNA complementary to the unique sequence of Fujinami sarcoma virus RNA, but minor differences were found by oligonucleotides fingerprinting. In the regions flanking the unique sequences, UR1 and Fujinami sarcoma viral RNAs contain distinct oligonucleotides, which are shared with oligonucleotides of the respective helper viral RNAs. Cell transformed with UR1 produce a single 29S RNA species which contains a UR1 unique sequence; this species is most likely the mRNA coding for the transforming protein. In UR1-transformed cells, a phosphoprotein fo 150,000 daltons (p150) was detected by immunoprecipitation with antiserum against gag proteins. p150 was associated with a protein kinase activity that was capable of phosphorylating p150 itself, immunoglobulin G of antiserum, and a soluble substrate, alpha-casein. This enzyme transferred phosphate exclusively to tyrosine residues of substrates in vitro, but p 150 labeled in vivo with 32P contained both phosphoserine and phosphotyrosine. The in vitro kinase reaction was not affected by the presence of cyclic AMP or cyclic GMP and strongly preferred Mn2+ over Mg2+. Thus, the properties of UR1 protein are almost identical to those of Fujinami sarcoma virus protein.
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PMID:Genetic structure, transforming sequence, and gene product of avian sarcoma virus UR1. 627 Mar 78

We have previously reported the crystallization of a mutant RNase T1(Y45W) with a synthetic modified trinucleotide ApGflpA [Hakoshima, T. et al. (1990) J. Biochem. 108, 695-698]. In the present report, we describe the crystal structure refined at 2.4 A resolution. During the refinement process, we found that the ApGflpA molecule was cleaved at the phosphodiester bond between the 5'-terminal adenosine and the subsequent 2'-fluoroguanosine. At the end of the refinement (R = 17.1%), it was supposed that the resulting molecules, i.e., 3'AMP and GflpA, were separately bound to the enzyme. In the complex structure, the binding-site of the enzyme was occupied by the guanine base of GflpA via a similar interaction to that of the enzyme complexed with 2'GMP, while the phosphate group of GflpA was not bound to the active site. The guanosine adopted the anti orientation on the glycosyl torsion angle with a C2'-endo-C3'-exo sugar pucker. This conformation resulted in the phosphate group protruding from the active site. The phosphate group of 3'AMP was bound to the active site of the enzyme and oriented itself toward the solvent region. This orientation was different from that of 2'AMP bound to the RNase T1(Y45W).
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PMID:Crystal structure of RNase T1(Y45W) complexed with 3'AMP and GflpA. 813 41

The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for initiation of protein synthesis in Escherichia coli. In attempts to identify regions of MTF that come close to the 3'-end of the tRNA, we oxidized 32P-3'-end-labeled E. coli initiator methionine tRNA with sodium metaperiodate and cross-linked it to MTF. The cross-linked MTF was separated from uncross-linked MTF by DEAE-cellulose chromatography, and the tRNA in the cross-linked MTF was hydrolyzed with nuclease P1 and RNase T1, leaving behind an oxidized fragment of [32P]AMP attached to MTF. Trypsin digestion of the cross-linked MTF followed by high pressure liquid chromatography of the digest yielded two peaks of radioactive peptides, I* and II*. These peptides were characterized by N- and/or C-terminal sequencing and by matrix-assisted laser desorption ionization mass spectroscopy. Peptide I* contained amino acids Gln186-Lys210 with Lys207 as the site of the cross-link. Peptide II*, a partial digestion product, contained amino acids Gln186-Arg214 also with Lys207 as the site of the cross-link. The molecular masses of peptides I* and II* indicate that the final product of the cross-linking reaction between the periodate-oxidized AMP moiety of the tRNA and Lys207 is most likely a morpholino derivative rather than a reduced Schiff's base.
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PMID:Lysine 207 as the site of cross-linking between the 3'-end of Escherichia coli initiator tRNA and methionyl-tRNA formyltransferase. 903 Jun 4

1. In order to understand the differences in pH optima and reaction rates of RNase A towards low molecular weight substrates and polymer substrates, the subsite structure of bovine pancreatic RNase A was studied. The kinetic studies of various sizes of oligouridylic acids showed that the size of the subsite is three nucleotides long. The kinetic studies on the inhibition of pUp, X-ray crystallographies of RNase A-ApC and pTp complexes, 31P-NMR studies on the binding of RNase A-pAp, and pTp showed the presence of P0, P2 and B3 sites. The location of the P0 site was assigned to be Lys66 by X-ray crystallography of the RNase A-pTp complex. The location of the P2 and/or P3/B3 site was determined by studying the enzymatic activities of several S-peptide analogs in which N-Leu was substituted for Lys7 and/or Lys1 coupled with S-protein toward various chain lengths of oligouridylic acids. The experiment suggested that P2 is Lys7 and P3/B3 is Lys1. 2. Several new pyrimidine base specific RNases were isolated and their primary structures were determined. They were two non-secretory RNases, a bovine liver alkaline RNase, a bovine brain RNase, and a bullfrog liver RNase. The bovine brain RNase has extra 16 amino acids at the C-terminus with O-glycosylated Ser. The bullfrog liver RNase was an extremely heat-stable RNase so far known. 3. Two new RNases belonging to RNase T1 family were isolated and their primary structures were elucidated. They were RNases isolated from Aspergillus saitoi and a mushroom (hiratake). The former RNase has a similar structure to RNase T1, but it was a base non-specific and guanylic acid preferential enzyme. From the results of X-crystallographic studies of this RNase, we suggested that the mechanism of RNase T1 RNase is essentialy a general acid-base catalysis between His40 and Glu58. 4. We isolated several fungal, plant and animal base non-specific acid RNases with a molecular mass about 24 kDa or more, and elucidated their primary structures. These RNases contain two sequences containing common 7-8 amino acid residues in common which include most of the amino acid residues important for the catalysis. Therefore, we proposed to designate these RNases as RNase T2 family RNase. On the basis of chemical modifications, kinetic studies and protein engineering studies of RNase Rh from Rhizopus niveus and RNase M from A. saitoi, we assigned that the catalytic site of RNase Rh consists of His46, His104, His109, Glu105, and Lys108. In the mechanism we proposed for RNase Rh, His46 and His109 work as a general acid and base catalysts. His104 was a phosphate binding site, and Glu105 and Lys108 might work to polarize a P=O bond of the substrate or stabilize the pentacovalent intermediate. However, in the reverse reaction of the transfer reaction step and the hydrolysis step of RNase Rh, His109 and His46 work as an acid and base catalyst, respectively. The X-ray crystallographic studies of RNase Rh, an RNase Rh-2'-AMP or d(ApC)complex, and the protein engineering studies of several mutant enzymes assigned the components of the major base recognition site (B1 site) and the minor base recognition site (B2 sites) of RNase Rh. The enzymatic studies of several mutant enzymes indicated that (i) Asp51 is very crucial for adenine base recognition, and the replacement of Asp51 by other amino acid, such as Thr, Ser, Glu, Asn makes RNase Rh more guanylic acid preferential, (ii) the replacement of Trp49 by Phe, and Tyr57 by Trp make the enzyme more pyrimidine and purine bases preferential, respectively. These trials are the first example of marked artificial change in the base specificity of RNases.
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PMID:[Structures and functions of ribonucleases]. 935 26

To isolate DNA for nucleoside analog incorporation studies, many investigators use RNase A to remove RNA from total cellular nucleic acid. We observed persistence of ribonucleotides from RNA in nucleic acid samples treated with RNase A alone. Although incubation of [5-3H]uridine-labeled nucleic acid with 50 microg/ml RNase A decreased tritium by 97%, HPLC analysis of the resulting DNA preparation digested to nucleosides revealed high levels of ribonucleosides. Increasing RNase A 10-fold (500 microg/ml) effected only a 1.7-fold reduction in ribonucleosides. Overall, the level of ribonucleosides was one-fourth that of the deoxynucleosides, primarily due to the high levels of guanosine. It was hypothesized that the ribonucleosides originated from guanosine-rich tracts of RNA since RNase A cuts preferentially 3' to pyrimidine monophosphates and to some extent after AMP. The addition of 0.05 microg/ml RNase T1, which preferentially cleaves RNA 3' to GMP, decreased total ribonucleosides by nearly 20-fold. In conclusion, we have developed a rapid method which removes greater then 99% of cellular RNA from nucleic acid extracts and a reversed-phase HPLC procedure that detects RNA contamination more sensitively than [5-3H]uridine labeling. These methods are useful for the determination of analog incorporation into DNA, especially for agents which incorporate into both DNA and RNA.
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PMID:Improved method to prepare RNA-free DNA from mammalian cells. 944 56


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