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Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bovine pancreatic ribonuclease is a DNA "melting" protein, since it binds with greater overall affinity to the single-stranded than to the double-stranded form of natural and synthetic deoxyribose-containing polynucleotides. As such, the DNA-RNase system provides a simple model for the more complex and biologically relevant melting protein-nucleic acid systems. Aspects of the DNA-RNase interactions which are related to the quantitative assessment of this system as a melting protein model are investigated here. A boundary sedimentation velocity technique is used to measure thermodynamic parameters of the interaction; association constants (Kh and Kc) and site sizes (nh and nc) are determined for the interaction of ribonuclease with native (double helical) and denatured (random coil) DNA. It is shown that log Kh and log Kc are linear functions of log [Na+], binding decreasing with increasing Na+ concentration, with Kh about 2 orders of magnitude smaller than Kc at the ionic strengths studied, nh and nc are approximately 8 and approximately 11 nucleotide residues, respectively, indicating that potential binding sites overlap. Binding to both forms of DNA is non-cooperative. It is shown by CD and ultraviolet spectroscopy that the binding of RNase to single- and double-stranded DNA perturbs the conformations of these polynucleotide conformations very little relative to the unliganded structures. Hydrodynamic methods are used to show that RNase binds to native DNA without altering the overall solution structure of the latter; however conditons which permit binding to, and stabilization of, transiently exposed single-stranded sequences result in a collapse of the stiff native DNA structure. We demonstrate by melting transition studies that ribonuclease does bring about an equilibrium destabilization of native DNA and poly [d(A-T)] and, by applying a ligand-perturbed helic in equilibrium coil theory developed by McGhee (McGhee, J.D. (1976) Biopolymers 15, 1345-1375), it is shown that the extent of the observed destabilization is in semiquantitative accord with expectations based on the measured affinity constants and site sizes for RNase binding to both DNA conformations. Spectral methods are used to show that the relative stability of native DNA sequences of varying base composition is the same in the presence and absence of ribonuclease, strongly arguing that this "melting" ligand "traps" single-stranded sequences transiently exposed by thermal fluctuations. RNase also undergoes an order in equilibrium disorder conformational transition as a function of temperature (the denatured form of RNase stabilizes native DNA, while native RNase destabilizes the native double helix), and the coupled equilibria involved in these interacting conformational changes are interpreted and discussed as possible models of genome regulatory interactions.
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PMID:DNA "melting" proteins. I. Effects of bovine pancreatic ribonuclease binding on the conformation and stability of DNA. 99 11

The terminal stage of differentiation of nucleated chicken erythrocytes is associated with an overall gene repression and a condensation of the repressed chromatin portion. Two-dimensional DNP electrophoresis has been used to separate transcriptionally active and repressed chromatin of mature chicken erythrocytes. The repressed chromatin fraction is shown to be enriched with histone H5 as well as with a 42-kDa nonhistone chromosomal protein. The 42-kDa protein designated here as MENT (mature erythrocyte nuclear termination stage-specific protein) is hyperexpressed at the terminal stage of chicken erythropoiesis and is accumulated in adult chicken erythrocyte nuclei. This protein was purified by ion-exchange chromatography from 0.4 M NaCl extracts of the erythrocyte nuclei. It appeared to be a basic polypeptide (pI 9.2) which, however, precipitated at low pH. When reconstituted in vitro with immature erythrocyte nuclei, MENT promoted condensation of intact nuclear chromatin and enhanced the solubilization of nuclease-digested polynucleosomes, thus mimicking the processes occurring in vivo at the final stage of erythrocyte maturation. The extent of dissociation of specific gene sequences from the nuclear matrix in MENT-treated nuclei is in striking correlation with their transcriptional activity. No other basic proteins (H5, cytochrome c, RNase A) added to the nuclear preparation at the same level as MENT (protein/DNA = 0.005) caused any effect on nuclear organization. No alterations were observed when MENT was mixed with erythroblasts and nonerythroid nuclei having little or no histone H5. We propose that MENT cooperates with histone H5 to complete the nuclear collapse in mature nucleated erythrocytes.
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PMID:A novel nonhistone protein (MENT) promotes nuclear collapse at the terminal stage of avian erythropoiesis. 172 33

The results presented here indicate that there are two slowly exchanging conformational isomers in unfolded bovine pancreatic ribonuclease A (RNase A) in the vicinity of Lys-41. The conformational heterogeneity is not observed in the fully folded protein. Therefore, one of the isomers may correspond to one of the slow-folding forms of the protein observed when refolding is initiated. These results were obtained from a chemically modified form of the protein, CL(7-41) RNase A, that has a dinitrophenyl cross-link between the epsilon-amino groups of Lys-7 and Lys-41. Extensive physical studies have shown that the cross-link does not significantly perturb the structure or the folding pathways of the protein. Therefore, the results obtained from this modified form of the protein are relevant to intact RNase A. The one-dimensional (1D) NMR spectrum of heat-unfolded CL(7-41) RNase A reveals that the singlet resonance for the C3H ring proton of the dinitrophenyl cross-link has been split into two unequal peaks in a 3:1 ratio, indicating that there are two distinct environments for the dinitrophenyl group. Variations in temperature, and the addition of urea, do not affect the relative peak intensities. The two peaks collapse into one after the protein is refolded. The observed splitting must originate from a slow reversible isomerization (greater than 100 msec) in a neighboring bond. The two most likely candidates are either the cis/trans isomerization of the Lys-41-Pro-42 peptide bond or hindered rotation about the disulfide bond between Cys-40 and Cys-95.
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PMID:Identification of a new site of conformational heterogeneity in unfolded ribonuclease A. 196 87

The maintenance of normal chromatin morphology requires ongoing RNA synthesis. We have examined the role of RNA in chromatin organization, using selective detergent extraction of cells, RNA synthesis inhibitors, and enzymatic digestion of nuclear RNA. Comparison of extracted and unextracted cells showed that the important features of chromatin architecture were largely unchanged by the extraction procedure. Normally, chromatin was distributed in small heterochromatic regions and dispersed euchromatic strands. Ribonucleoprotein granules were dispersed throughout the euchromatic regions. Exposure to actinomycin led to the redistribution of chromatin into large clumps, leaving large empty spaces and a dense clustering of the remaining ribonucleoprotein granules. When the nuclei of extracted cells were digested with RNase A, there was a rearrangement of chromatin similar to but more pronounced than that seen in cells exposed to actinomycin. The inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole also inhibits RNA synthesis but by a different mechanism that leaves no nascent RNA chains. The drug had little effect on chromatin after brief exposure but resembled actinomycin in its effect at longer times. We also examined the structure of the nuclear matrix to which most heteronuclear RNA remains associated. Pretreatment of cells with actinomycin or digestion of the nuclear matrix with RNase A caused the matrix fibers to collapse and aggregate. The experiments show a parallel decay of chromatin and of nuclear matrix organization with the depletion of nuclear RNA and suggest that RNA is a structural component of the nuclear matrix, which in turn may organize the higher order structure of chromatin.
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PMID:Chromatin architecture and nuclear RNA. 291 67

The kinetics of unfolding and refolding of porcine ribonuclease were investigated. The unfolded state of this protein was found to consist of a fast-refolding species (UF) and two slow-refolding species (UIS and UIIS). After the rapid collapse of the structure during the N (native)----UF unfolding reaction, UIS and UIIS are produced from UF by two independent slow isomerizations of the unfolded polypeptide chain, leading ultimately to a mixture of about 10% UF, 20% UIIS and 70% UIS molecules at equilibrium. This is at variance with all other ribonucleases investigated to date, which show a distribution of 20% UF, 60 to 70% UIIS and only 10 to 20% UIS. The two isomerizations of the unfolded porcine protein differ strongly in rate. The first process with tau = 250 seconds (10 degrees C) leads to an increase in the fluorescence of Tyr92 and was identified as cis in equilibrium trans isomerization of Pro93. At equilibrium, most unfolded molecules contain an incorrect trans Pro93. The second isomerization is much slower (tau = 1300 s at 10 degrees C) and leads to a predominance of the incorrect isomer as well. Like isomerization of Pro93, it is governed by an activation enthalpy of 22 kcal/mol (92 kJ/mol) and it was tentatively assigned to the Pro114-Pro115 sequence of porcine ribonuclease. Because of the wide separation in rate between the two reactions, molecules with an incorrect isomer only at Pro93 accumulate transiently after unfolding. These are the UIIS molecules. Most of them are finally converted to UIS by the 1300 second process. All molecules that have undergone this isomerization refold very slowly, i.e. the UIS molecules. The major fraction contains two incorrect isomers. A 1300 second isomerization after unfolding and a predominant very slow refolding reaction were observed only for the porcine protein. We suggest that these changes in the folding mechanism may be correlated with the presence of the Pro114-Pro115 sequence, which occurs only in porcine ribonuclease. The refolding pathway of porcine UIIS involves the rapid formation of a native-like intermediate with an incorrect trans Pro93 as was found previously for the bovine ribonuclease, where the UIIS species predominates in the unfolded state.
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PMID:Folding mechanism of porcine ribonuclease. 380 74

An electron microscopy study has been made of the effects of dissolution of the plasma membrane of Escherichia coli with sodium dodecyl sulfate (SDS) on the organization of the nucleoplasm and the cytoplasm. The alterations observed in time course experiments were related to absorbance changes and to release of macromolecules from the cells. As the cells became plasmolyzed, under the conditions used, the first visible effect of SDS was a collapse of the plasmolysis spaces. This was accompanied by a displacement of the nuclear material which then appeared in broad contact with the redeployed plasma membrane. This initial displacement of nuclear material to the cell border may indicate an association between the nucleoplasm and the plasma membrane. Upon further dissolution of the plasma membrane, the nuclear material receded from the cell margin and contracted into an axial filament. Meanwhile, the cytoplasm dissociated into an amorphous, Pronase-sensitive component and an electron-opaque, granular one sensitive to ribonuclease. The latter represented one continuous area of ribosomal structures surrounding the nucleoplasm, an organization which did not occur when the cells were inhibited with rifamycin before SDS treatment. During prolonged SDS interaction, approximately 65% of the cellular protein, 25% of the ribonucleic acid and 40% of the deoxyribonucleic acid were released from the cells concomitant with the disappearance of the amorphous cytoplasmic part, expansion of the ribosomal aggregate, and rearrangement of the nuclear material at the cell periphery. The observations support the contention that all ribosomal structures bear a direct relationship with the nucleoplasm.
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PMID:Effects of treatment with sodium dodecyl sulfate on the ultrastructure of Escherichia coli. 455 30

Aldehyde-fixed rat tissues were variously dehydrated and impregnated in water-miscible 2-hydroxypropyl methacrylate (HPMA) containing 3 to 20 per cent water and 0.1 per cent alpha,alpha-azobisisobutyronitrile as catalyst for subsequent polymerization with ultraviolet light. Heat polymerization was also effective. Blocks of embedded tissue readily gave ultrathin sections, which required staining by uranyl acetate and/or lead stains to give adequate contrast for electron microscopy. The ultrastructure of pancreas, kidney, muscle, and intestine was well preserved by aldehyde fixation alone. Use of postfixation in osmium tetroxide or direct osmium tetroxide fixation was unsatisfactory. The fine structure of aldehyde-fixed liver from fasted rats was well preserved, whereas that from normal rats showed considerable disorganization and collapse, apparently because of extraction of glycogen during the embedding procedure. Enzymatic extraction of proteins by pepsin and of ribonucleic acid by ribonuclease after either formaldehyde or glutaraldehyde fixation was rapidly effected by direct treatment of ultrathin sections with solutions of the enzymes. In contrast, no digestion of chromatin by deoxyribonuclease could be detected. In spite of this present limitation, HPMA appears to have several advantages over earlier water-miscible embedding media for electron microscopy and to be particularly suitable for ultrastructural cytochemistry.
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PMID:Hydroxypropyl methacrylate, a new water-miscible embedding medium for electron microscopy. 585 16

A cytochemical and electron microscope study has been made of leaves of sugar beet infected with beet yellows virus. Inclusions of particles, which agree in size with beet yellows virus particles isolated by other investigators, have been localized in the ground cytoplasm, in the chloroplasts, and in the nuclei. These particles are circa 100 A in diameter and have an electron-transparent core of 30 to 40 A. Use of acridine orange, azure B, and pyronine Y has revealed that the cytoplasmic inclusion bodies, which consist wholly of the elongate particles, have a strong RNA reaction removable by RNase pretreatment. Particles observed in the chloroplasts may or may not be associated with lipid spheres. If they are, the particles are confined to the periphery of the spheres. In this position the particles are arranged tangentially and are further arranged parallel into groups which lie at various angles to one another. Within the groups the particles are regularly spaced in a three dimensional lattice. Particles located free in the stromal regions are often arranged regularly in curved rows which lie parallel to one another so that a three dimensional lattice is formed. The dispersed and compact forms of virus inclusions are described and related to the condition of the associated cytoplasm. The ground cytoplasm of cells associated with the sieve elements contains numerous ribosomes. A decrease in the number of ribosomes is concomitant with the increase in size of virus aggregations in a cell. Vesiculation of some component of the cytoplasm occurs during the period of virus replication. The vesicles are approximately 100 mmicro in diameter and could be derived from the dictyosomes. At later stages of infection these vesicles collapse and convoluted membranous material appears.
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PMID:Ultrastructural features of Beta leaves infected with beet yellows virus. 597 43

Heat shock induces changes in G1 CHO cell nuclear matrix (NM) ultrastructure that may be related to heat-induced nuclear protein accumulation (Wachsberger and Coss, 1993, J. Cell. Physiol., 155:615-634). The present study quantitates recovery of alterations in NM fine structure in CHO cells heated in G1 and compares structural recovery with recovery of bulk RNA synthesis and surviving fraction (SF). Morphology of NM preparations was quantified 30 min and 20 hr following heat shock by 1) measurement of the number of fiber anastomosing points per unit area per NM, and 2) measurement of the length of fibers between points of anastomoses within individual NMs. Architectural recovery was nearly complete within 20 hr in cells heated at 43 degrees C or 45 degrees C with SFs of 0.27 or greater. No recovery of architecture was observed in heated cells with SFs of approximately 0.01 or less. The residual damage to NMs was associated with RNA-containing fiber networks as determined by means of RNase gold labeling. Recovery from inhibition of RNA synthesis following heat shock was related to recovery of NM architecture. It is suggested that 1) repair of NM architecture does not require full recovery of bulk RNA synthesis, and 2) partial or complete irreversible collapse of the NM may be responsible, in part, for heat-induced, interphase cell death.
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PMID:Recovery of nuclear matrix ultrastructure of interphase CHO cells after heat shock. 751 8

Hydrogen-deuterium exchange of 39 amide protons of Bacillus amyloliquefaciens ribonuclease (barnase) was analyzed by two-dimensional nuclear magnetic resonance in the presence of micromolar concentrations of the molecular chaperones GroEL and SecB. Both chaperones bound to native barnase under physiological conditions and catalyzed exchange of deeply buried amide protons with solvent. Such exchange required complete unfolding of barnase, which occurred in the complex with the chaperones. Subsequent collapse of unfolded barnase to the exchange-protected folding intermediate was markedly slowed in the presence of GroEL or SecB. Thus, both chaperones have the potential to correct misfolding in proteins by annealing.
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PMID:Catalysis of amide proton exchange by the molecular chaperones GroEL and SecB. 857 Nov 25


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