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:3.1.27.4 (ribonuclease)
6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When closed circular SV40 DNA containing 58 negative superhelical turns is used as a template for RNA synthesis with Escherichia coli RNA polymerase, a fraction of the RNA product remains complexed with the DNA. The RNA in the complex is resistant to ribonuclease in high salt, and the Tm indicates that it is hydrogen bonded to the DNA. The mole ratio of RNA to DNA nucleotides in the complex ranges from 0.01 to 0.08; the RNA ranges in length from 80 to 600 nucleotides. The formation of the complex is dependent on the circular DNA being topologically underwound since no complex is formed when closed circular DNA containing zero superhelical turns is used as the template. The DNA-RNA complex can serve as a primer-template combination for in vitro DNA synthesis by E. coli DNA polymerase I. After synthesis with (alpha-32P)-labeled deoxyribonucleoside triphosphates followed by alkaline hydrolysis, the isolation of 32P-labeled ribonucleotides is evidence for a covalent linkage between the RNA and the DNA synthesized. During the in vitro DNA synthesis, the template is nicked at a low rate, and the nicked molecules support extensive DNA synthesis. This observation indicates that only limited synthesis can occur on unnicked molecules possibly owing to the topological constraints against unwinding of the helix. Possible models for in vivo priming of double-stranded DNA by E. coli RNA polymerase are discussed.
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PMID:Priming of superhelical SV40 DNA by Escherichia coli RNA polymerase for in vitro DNA synthesis. 16 2

The microenvironment of histidine-48 of bovine pancreatic ribonuclease A was investigated by proton magnetic resonance spectroscopy (1H NMR) using partially deuterated enzyme in which resolution of the C(2)-H resonance of histidine-48 was simplified. The NMR titration curves at 100 and 250 MHz of histidine-48 of ribonuclease A are discontinuous both for the enzyme alone in 0.3 M chloride and for its complex with cytidine 3'-phosphate. This suggests that titration of histidine-48 occurs only as the result of a slow conformational transition. The sum of the peaks corresponding to histidine-48 in the acid-stable and base-stable forms of the enzyme is less than one proton in the transition region, which indicates that there exists at least one intermediate conformational form of the enzyme. The transition from the acid-stable form to an intermediate form has a pHmid of 5.6, and the transition from an intermediate form to the base-stable form has a pHmid of 6.9. In ribonuclease S and in ribonuclease A in the presence of 0.3 M acetate, the titration curve of histidine-48 is continuous, and the area of the peak is uniform throughout the titration. Proton NMR difference spectra at 100 and 250 MHz reveal a pH-induced conformational change with a pHmid of 5.7 that affects the chemical shift of a single tyrosine residue. This conformational transition is absent in ribonuclease S and is altered in ribonuclease A by the presence of either acetate or cytidine 3'-monophosphate. It is postulated that the same conformational transition is responsible for both the tyrosine perturbation and the disappearance of the histidine-48 peak observed in the acid-stable form of the enzyme. It is proposed that the perturbed tyrosine is tyrosine-25. The transition with pHmid 5.6 is attributed to dissociation of aspartic acid-14, and the transition with pHmid 6.9 is assigned to dissociation of histidine-48. A peak in the aromatic region that moves upfield on addition of the competitive inhibitor cytidine 3'-monophosphate is assigned to a tyrosine, and evidence is presented that this tyrosine is tyrosine-25. Inhibitor binding appears to induce a conformational change in the histidine-48/tyrosine-25 region which is remote from the active site.
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PMID:Correlation proton magnetic resonance studies at 250 MHz of bovine pancreatic ribonuclease. II. pH and inhibitor-induced conformational transitions affecting histidine-48 and one tyrosine residue of ribonuclease A. 24 Mar 91

Red kangaroo (Macropus rufus) ribonuclease was isolated from pancreatic tissue by affinity chromatography. The amino acid sequence was determined by automatic sequencing of overlapping large fragments and by analysis of shorter peptides obtained by digestion with a number of proteolytic enzymes. The polypeptide chain consists of 122 amino acid residues. Compared to other ribonucleases, the N-terminal residue and residue 114 are deleted. In other pancreatic ribonucleases position 114 is occupied by a cis proline residue in an external loop at the surface of the molecule. Other remarkable substitutions are the presence of a tyrosine residue at position 123 instead of a serine which forms a hydrogen bond with the pyrimidine ring of a nucleotide substrate, and a number of hydrophobichydrophilic interchanges in the sequence 51-55, which forms part of an alpha-helix in bovine ribonuclease and exhibits few substitutions in the placental mammals. Kangaroo ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64). The enzyme differs at about 35-40% of the positions from all other mammalian pancreatic ribonucleases sequenced to date, which is in agreement with the early divergence between the marsupials and the placental mammals. From fragmentary data a tentative sequence of red-necked wallaby (Macropus rufogriseus) pancreatic ribonuclease has been derived. Eight differences with the kangaroo sequence were found.
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PMID:The amino-acid sequence of kangaroo pancreatic ribonuclease. 65 39

The complex between ribosomal protein L24 and its RNA binding site (that region of the 23S RNA which the protein protects from ribonuclease digestion) has been studied by various physicochemical methods. The RNA is composed of two fragments of about 160 and 140 nucleotides which interact with each other to form the L24 binding site. Circular dichroism spectroscopy suggests that the two interacting fragments have a unique region of secondary structure which is not present in either of the two components alone; hence there are important structural interactions between regions of the RNA which are separated in the primary sequence. Addition of the L24 protein to the RNA site promotes a structural change associated with base unstacking, but with little or no change in the hydrogen-bonded base pairing. Heat activation is not required for complex formation. Thermal denaturation studies reveal a broad featureless transition and the amount of hypochromic change indicates that the RNA site contains less secondary structure than other RNAs such as tRNA and total rRNA. Temperature-jump relaxation measurements on the mechanism of unfolding of the RNA show a concerted melting of the entire secondary and tertiary structure, which is altered upon addition of the protein. A structrual basis for this RNA-protein complex is discussed.
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PMID:Physical characterization of a ribosomal nucleoprotein complex. 78 77

In terms of the mechanical model of molecules, a calculation has been carried out of possible positions and binding energies of 1-methyl uracyl in the contact region of the ribonuclease S active site. In the most preferential orientation, 1-methyl uracyl forms hydrogen bonds C(2)=O(uracyl)...H-N(Thr-45), N-H...Ogamma (Thr-45), C(4)=O... ...H-Ogamma (Ser-123). The base position found (atom coordinates are given) is in complete qualitative agreement with the position of the uracyl in UpcA bound to ribonuclease S as revealed by X-ray analysis. The influence studied of methyl substitution in positions 3 and 5 of the pyrimidine cycle on the base orientation within the protein field. It has been shown that the formation of hydrogen bonds with Thr-45 and Ser-123 is not prerequisite for productive fixation of the phosphoribosyl nucleotide moiety in the catalytic region of the enzyme active site.
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PMID:[A theoretical analysis of the binding of methyl derivatives of uracil at the contact portion of the active center of ribonuclease S]. 94 May 57

Circular dichroism (CD) in the 240-300-nm region was used to study the conformation of DNA and RNA complexed with proteins in isolated nucleoli form HeLa cells. Deoxyribonuclease or ribonuclease digestion was employed to obtain (1) the individual CD spectra of nucleolar DNA or RNA in complex form with proteins, or in free form; and (2) the experimental CD baseline correction to exclude contributions from nonnucleic acid sources such as light scattering artifacts and proteins. The CD spectrum of nucleolar DNA in DNA-protein complexes was highly reduced in ellipticity in comparison with protein-free DNA. It showed a positive peak at 283 nm with a molar ellipticity [theta]283 = 1200 deg cm2 dmol-1 and a crossover at 262 nm. Addition of sodium dodecylsulfate shifted the peak to 276 nm with [theta]276 8000 deg cm2 dmol-1 and a crossover at 254 nm. The CD spectrum of nucleolar RNA in RNA-protein complexes was also reduced in comparison with protein-free RNA, showing a peak at 269 nm ([theta]269 = 6900 deg cm2 dmol-1), and a crossover at 250 nm. Addition of sodium dodecyl sulfate shifted the peak to 265 nm with [theta]265 = 18 000 deg cm2 dmol-1 and a crossover at 246 nm. The low ellipticity of both nucleolar DNA and RNA when complexed with proteins was increased by treatment with sodium chloride, urea, or heparin. This suggests that some ionic, hydrophobic, and hydrogen bondings are involved in the nucleic acid-protein interaction in nucleolar chromatin similar to that observed in nuclear chromatin.
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PMID:Circular dichroic studies of the DNA and RNA of nucleoli. 94 79

The COOH-terminal tetradecapeptide of ribonuclease A, Glu-Gly-Asn-Pro-Tyr-Val-Pro-Val-His-Phe-Asp-Ala-Ser-Val, and two analogs, [Ser(Me)-123]-RNase 111-124 and [Ala-123]-RNase 111-124, were synthesized by the solid phase method and were purified to chromatographic and electrophoretic homogeneity. Methods are described for the hydrolysis and quantitative amino acid analysis of peptides containing O-methylserine. The peptides were combined noncovalently with RNase 1-118 and examined for ability to regenerate enzymatic activity in the presence of the substrates C greater than p, U greater than p, poly(C) poly(U), and poly(AF). The dissociation constants of the peptide-protein complexes, and the Michaelis constants for C greater than p and U greater than p with the reconstituted enzymes were determined. The data were used to test hypotheses, drawn from x-ray crystallographic and other studies, for the role of serine-123 in the binding of substrates by ribonuclease. It was found that Ser-123- and Ala-123-containing peptides were equally active for the hydrolysis step when measured with C greater than p as substrate and for the transphosphorylation step as measured in the assays with poly(C). The serine and alanine analogs were also equally active for the transphosphorylation step when poly AF was the substrate. With U greater than p as substrate the alanine analog was 4 times less active than the serine derivative and with poly U it was 2 times less active. The semisynthetic enzyme composed of RNase 1-118 and [Ala-123]-RNase 111-124, therefore, shows appreciable selectivity for substrates containing cytosine. It was concluded that a hydrogen bond between the hydroxyl of serine-123 and the C4 amino group of cytidine or the C-7 amino group of formycin is not important for substrate binding and catalytic activity. In contrast, the hydrogen bond between the hydroxyl of serine 123 and the C-4 carbonyl oxygen of uridine contributes significantly to substrate binding and catalytic activity. The data with serine-O-methyl ether at position 123 in the tetradecapeptide were less clear because it was difficult to separate steric effects from the contributions of hydrogen bonding. Substrate binding to ribonuclease was rationalized in terms of a binding energy equivalent to a total of two hydrogen bonds per pyrimidine.
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PMID:The role of serine-123 in the activity and specificity of ribonuclease. Reactivation of ribonuclease 1-118 by the synthetic COOH-terminal tetradecapeptide, ribonuclease 111-124, and its O-methylserine and alanine analogs. 111 2

The formation of hydrogen-bonded structure in the folding reaction of ubiquitin, a small cytoplasmic protein with an extended beta-sheet and an alpha-helix surrounding a pronounced hydrophobic core, has been investigated by hydrogen-deuterium exchange labeling in conjunction with rapid mixing methods and two-dimensional NMR analysis. The time course of protection from exchange has been measured for 26 back-bone amide protons that form stable hydrogen bonds upon refolding and exchange slowly under native conditions. Amide protons in the beta-sheet and the alpha-helix, as well as protons involved in hydrogen bonds at the helix/sheet interface, become 80% protected in an initial 8-ms folding phase, indicating that the two elements of secondary structure form and associate in a common cooperative folding event. Somewhat slower protection rates for residues 59, 61, and 69 provide evidence for the subsequent stabilization of a surface loop. Most probes also exhibit two minor phases with time constants of about 100 ms and 10 s. Only two of the observed residues, Gln-41 and Arg-42, display significant slow folding phases, with amplitudes of 37% and 22%, respectively, which can be attributed to native-like folding intermediates containing cis peptide bonds for Pro-37 and/or Pro-38. Compared with other proteins studied by pulse labeling, including cytochrome c, ribonuclease, and barnase, the initial formation of hydrogen-bonded structure in ubiquitin occurs at a more rapid rate and slow-folding species are less prominent.
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PMID:Early hydrogen-bonding events in the folding reaction of ubiquitin. 131 11

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.
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PMID:RNase T1 mutant Glu46Gln binds the inhibitors 2'GMP and 2'AMP at the 3' subsite. 135 Jun 42

Seven hydrophobic residues ranging in size from glycine to phenylalanine have been substituted for the wild-type methionine residue at position 13 in a 15-residue truncated version (S15) of S-peptide, the small component of ribonuclease S. Complexes of both S-15 and the seven variants with S-protein yielded isomorphous crystals. The structures of all eight complexes have been refined to final R-factors in the range of 17-19%. [See Kim, E. E. Varadarajan, R., Wyckoff, H. W., and Richards, F. M. (1992) Biochemistry (preceding paper in this issue) for the description of the reference S-15 complex.] Multiple side-chain conformations were seen for six residues in all of the complexes and for two to three additional residues in at least some of the complexes. Three of the complexes, Gly, Ala, and alpha-amino-n-butyric acid (ANB), contained a single water molecule in the cavity near residue 13 that makes three hydrogen bonds to protein atoms. Although space is available, no evidence for additional water in this region, ordered or disordered, was found. The atoms in the cavity wall tend to shrink the cavity by moving in on the small residues and to swell the cavity by moving out for the larger Phe substitution. A swelling seen with leucine was attributed to a shape effect since Leu, Ile, and Met all have the same volume. A slight volume contraction of the collection of interior residues outside of the region of position 13 was also noted. (All changes noted are in the direction to maintain a constant packing density averaged over the whole protein.) Leu51, a surface hydrophobic residue, moved considerably in the G, A, and ANB complexes in directionswhich would tend to decrease the cavity volume. The only other major change in position, 1.5 A, was the 66-69 loop, which is about 25 A from position 13. His12, Phe120, and Asp121 appear to be involved in this movement, but the connection with position 13 is not clear at all. The thermodynamic data on the association reaction for all of these complexes have been previously reported [Connelly, P. R., Varadarajan, R., Sturtevant, J. M., & Richards, F. M. (1990) Biochemistry 29, 6108-6114; Varadarajan, R., Connelly, P. R., Sturtevant, J. M., & Richards, F. M. (1992) Biochemistry 31, 1421-1426]. Some comments are offered on our initial attempts to correlate the structural changes with the changes in the thermodynamic parameters.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Crystallographic structures of ribonuclease S variants with nonpolar substitution at position 13: packing and cavities. 146 20


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