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 mother enzyme of RNase T1 was co-crystallized with its natural product, 3'-GMP at pH 4.0. The X-ray structure of this complex was refined with 2432 reflections in the 5.4-2.6 A range using a stereochemical restrained method (conventional R = 27.4%). The overall polypeptide chain folding is very similar in the secondary structure elements to the RNase T1 in the complex with 2'-GMP crystallized also at pH 4.0, but larger conformational changes occur in the loop regions. The base recognition scheme is identical in both complexes but in RNase T1 X 3'-GMP, the ribose phosphate is not seen in the electron density, probably due to static disorder.
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PMID:Three-dimensional structure of the ribonuclease T1 X 3'-guanylic acid complex at 2.6 A resolution. 298 48

The biochemical properties of a virulent and an attenuated strain of foot-and-mouth disease virus (FMDV) Type 0(1) Campos (0(1)C) were compared in order to establish differences that could account for their altered biological functions. The avirulent strain (0(1)C-O/E) was derived from the virulent strain 0(1)C by serial passages in chicken embryos. Analysis of the RNase T1-generated oligonucleotides of the viral RNA through one- and two-dimensional (2D) gel electrophoresis (fingerprints) revealed a few changes in the genome structure of the 0(1)C-O/E strain compared to the wild type strain. In addition there was a significant decrease in the length of the poly(C) rich tract of the 0(1)C-O/E RNA. All virion structural proteins, except VP4, their precursors, and the viral RNA polymerase (p56a) show charge differences. In addition a significant decrease in the apparent molecular weight of polypeptide p100 (primary translational product from the 3' end region of the genome) of the attenuated strain was observed.
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PMID:Biochemical characterization of an aphthovirus type 0(1) strain campos attenuated for cattle by serial passages in chicken embryos. 299 71

Guanidine resistance (gr) mutations of foot-and-mouth disease virus were mapped by recombining pairs of temperature-sensitive mutants belonging to different subtypes. In each cross, one parent possessed a gr mutation. Recombinants were isolated by selection at the nonpermissive temperature and assayed for the ability to grow in the presence of guanidine. From the progeny of three crosses, four different types of recombinant were distinguished on the basis of protein composition and RNA fingerprint. The sequences of the RNase T1-resistant oligonucleotides were determined and located in the full-length sequence of foot-and-mouth disease virus. The resulting maps show that (i) each recombinant was generated by a single genetic crossover, and (ii) both of the gr mutations studied were located within an internal 2.9-kilobase region which spans the P34 gene. This supports our hypothesis that guanidine inhibits the growth of foot-and-mouth disease virus by acting on nonstructural polypeptide P34. Additional evidence was provided by RNA fingerprinting gr mutants. In two of four cases the gr mutation was associated with a change in an oligonucleotide located near the 3' end of the P34 gene; in one of these the nucleotide substitution was identified.
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PMID:Recombination and oligonucleotide analysis of guanidine-resistant foot-and-mouth disease virus mutants. 299 45

Several major mRNA species of mouse and other mammalian cells occur both as small untranslated ribonucleoprotein particles and as functional molecules associated with ribosomes in polysomes. One of these, that codes for a 21-kDa polypeptide, was analyzed with respect to distribution of sites accessible to RNase T1 in the 5'-noncoding region. This region, which is about 100 nucleotides long, contains several sites that are highly sensitive to the enzyme, as well as many G residues not susceptible to cleavage. The distribution of highly sensitive sites was compared in the active and inactive states of the P21 mRNA present in cytoplasmic extracts by subjecting the extract to limited nuclease digestion followed by separation of partially fragmented polysomes from free messenger ribonucleoprotein particles. The mRNA in polysomes contained two highly sensitive sites, one near the 5' terminus and the other in the middle of the region, next to a sequence potentially capable of Shine-Dalgarno interaction. The untranslated molecules lacked the 5'-proximal site but had several highly accessible sites not present in the active molecules. The initiation AUG showed little accessibility both in polysomes and in messenger ribonucleoproteins. Both forms were quite different from the deproteinized mRNA with respect to distribution of nuclease-sensitive sites. Our results indicate that interaction of the mRNA with cytoplasmic factors strongly affects its conformation in the 5'-noncoding region and that a particular conformation may be important for effective interaction with ribosomal particles during polypeptide chain initiation.
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PMID:Shifts in configuration of the 5'-noncoding region of a mouse messenger RNA under translational control. 313 Mar 78

Recognition by ribonuclease T1 of guanine bases via multidentate hydrogen bonding and stacking interactions appears to be mediated mainly by a short peptide segment formed by one stretch of a heptapeptide, Tyr42-Asn43-Asn44-Tyr45-Glu46-Gly47- Phe48. The segment displays a unique folding of the polypeptide chain--consisting of a reverse turn, Asn44-Tyr45-Glu46-Gly47, stabilized by a hydrogen-bond network involving the side chain of Asn44, the main-chain atoms of Asn44, Gly47 and Phe48 and one water molecule. The segment is connected to the C terminus of a beta-strand and expands into a loop region between Asn43 and Ser54. Low values for the crystallographic thermal parameters of the segment indicate that the structure has a rigidity comparable to that of a beta-pleated sheet. Replacement of Asn44 with alanine leads to a far lower enzymatic activity and demonstrates that the side chain of Asn44 plays a key role in polypeptide folding in addition to a role in maintaining the segment structure. Substitution of Asn43 by alanine to remove a weak hydrogen bond to the guanine base destabilized the transition state of the complex by 6.3 kJ/mol at 37 degrees C. In contrast, mutation of Glu46 to alanine to remove a strong hydrogen bond to the guanine base caused a destabilization of the complex by 14.0 kJ/mol. A double-mutant enzyme with substitutions of Asn43 by a histidine and Asn44 by an aspartic acid, to reproduce the natural substitutions found in ribonuclease Ms, showed an activity and base specificity similar to that of the wild-type ribonuclease Ms. The segment therefore appears to be well conserved in several fungal ribonucleases.
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PMID:Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering. 315 Oct 17

The complete amino acid sequence of ribonuclease U1 (RNase U1), a guanine-specific ribonuclease from a fungus, Ustilago sphaerogena, was determined by conventional protein sequencing, using peptide fragments obtained by several enzymatic cleavages of the performic acid-oxidized protein. The oxidized protein was first cleaved by trypsin and the resulting peptides were purified and their amino acid sequences were determined. These tryptic peptides were aligned with the aid of overlapping peptides isolated from a chymotryptic digest of the oxidized protein. The amino acid sequence thus deduced was further confirmed by isolation and analysis of peptides obtained by digestion of the oxidized protein with lysyl endopeptidase. The location of the disulfide bonds was deduced by isolation and analysis of cystine-containing peptides from a chymotryptic digest of heat-denatured RNase U1. These results showed that the protein is composed of a single polypeptide chain of 105 amino acid residues cross-linked by two disulfide bonds, having a molecular weight of 11,235, and that the NH2-terminus is blocked by a pyroglutamate residue. It has an overall homology with other guanine-specific or related ribonucleases, and shows 48% identity with RNase T1 and 38% identity with RNase U2.
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PMID:The amino acid sequence of ribonuclease U1, a guanine-specific ribonuclease from the fungus Ustilago sphaerogena. 316 89

The calculation of induced dipole moments and of their contribution to electrostatic effects in proteins is implemented following the approach of Warshel. Isotropic polarizabilities are assigned to individual atoms, and the resulting deviation from pairwise interactions is treated by a self-consistent iterative procedure. We give a detailed description of how the formalism is implemented in molecular mechanics and molecular dynamics simulation procedures, and report results based on calculations performed on crystal structures of crambin, liver alcohol dehydrogenase and ribonuclease T1. We focus our analysis on evaluating the contribution of polarizability of the protein matrix to electrostatic energies, local fields, to dipole moments of peptide groups and of secondary structure elements in the polypeptide chain. Our calculations confirm that induced dipole moments in proteins provide important stabilizing contributions to electrostatic energies, and that these contributions cannot be mimicked by the usual approximations where either a continuum dielectric constant, or a distance-dependent dielectric function is used. We find that induced protein dipoles appreciably affect the magnitude and direction of local electrostatic fields in a manner that is strongly influenced by the microscopic environment in the protein. Most strongly affected are fields in charged groups that are involved in close interactions with other charged groups, while the influence on local fields of aliphatic groups is marginal. We find, moreover, that induction effects from surrounding protein atoms tend on average to increase peptide dipoles and helix macro-dipoles by about 16%, again reflecting electrostatic stabilization by the protein matrix, and show that (at least in the alpha/beta domain of alcohol dehydrogenase) the contribution of side-chains to this stabilization is significant.
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PMID:Calculations of electrostatic properties in proteins. Analysis of contributions from induced protein dipoles. 343 Jun 27

Poly(A)-protein particles were prepared from rat liver nuclear extract after digestion with pancreatic ribonuclease and ribonuclease T1 by sucrose gradient centrifugation. The particles were sedimented in a range of 9-23S with a peak at 16S. The particles isolated in this manner were 99-100% resistant to further pancreatic ribonuclease treatment and contained more than 90% adenylic acid. In CsCl density gradient the nuclear poly(A)-protein particles banded in a narrow density range of 1.28-1.32 g/cm3 with a peak at 1.30 g/cm3, which corresponds to about 90% of protein in the particles. The average length of the poly(A) molecules prepared from the 16-S particles was about 140 nucleotides. Urea/sodium dodecyl sulphate/polyacrylamide gel electrophoresis demonstrated two major polypeptide components with Mr of 63 000 and 90 000 and at least ten minor polypeptides in the 45 000-130 000-Mr range. In sodium dodecyl sulphate/polyacrylamide gels the 63 000-Mr polypeptide was the only one major component. Amino acid analysis of the polypeptides bound to nuclear poly(A) revealed that the polypeptides contained a relatively large amount of aspartic acid + asparagine and glutamic acid + glutamine (24%). Treatment of glutaraldehyde-fixed particles with micrococcal nuclease showed that more than 90% of the poly(A) was accessible to the enzyme, thus almost the entire poly(A) should be located on the surface of the particles. On the basis of the results a model for the 'average' 16-S particle was constructed.
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PMID:Structural characterization of nuclear poly(A)-protein particles in rat liver. 683 52

The major mRNA for adenovirus 2 polypeptide pVIII sediments at 18S as assayed by in vitro translation in the messenger-dependent rabbit reticulocyte lysate system. However, a small amount of messenger activity for pVIII sediments at abut 27S, coincident with the mRNA for 100 K. Isolation and fractionation of poly(A) containing RNA following in vitro translation of 27S 100 K mRNA demonstrated the appearance of an 18S messenger activity for pVIII, which is approximately the size of the authentic mRNA for this protein. Partial degradation of 27S 100 K mRNA with alkali or ribonuclease T1 also results in activation of an 18S messenger activity for pVIII suggesting that in vitro messenger activity for pVIII associated with 27S RNA is due to degradation of 100 K mRNA during translation in the cell-free system.
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PMID:Activation of an internal initiation site for protein synthesis during in vitro translation. 743 22

Alpha-sarcin is a cytotoxic protein composed of a single polypeptide chain. This protein shows a significant degree of amino acid sequence similarity with a group of several phylogenetically related fungal ribonucleases. The leading member of such a group is ribonuclease T1. Three proteins of this group, ribonucleases T1, Ms and F1, are well known in terms of their crystal structures. These data have been used to propose a conformation for alpha-sarcin. The secondary structure of the cytotoxin would contain one alpha-helix segment as well as around six beta-strands and 14 beta-turns. The folding of these structural motifs is proposed by comparison with the three-dimensional structure of the three proteins from the ribonuclease T1 subfamily. The four longest beta-strands of alpha-sarcin would define an antiparallel beta-sheet structure resulting in a highly hydrophobic domain. The predicted folding for alpha-sarcin is discussed in terms of the ability of this protein to electrostatically and hydrophobically interact with phospholipid vesicles. The proposed conformation would explain how a highly polar protein, such as alpha-sarcin, can produce membrane destabilization resulting in protein translocation across lipid bilayers.
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PMID:Predictive study of the conformation of the cytotoxic protein alpha-sarcin: a structural model to explain alpha-sarcin-membrane interaction. 771 96


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