Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.27.4 (ribonuclease)
 6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two-dimensional (2D) 1H NMR experiments using deuterium labeling have been carried out to investigate the solution of ribonuclease HI (RNase HI) from Escherichia coli (E. coli), which consists of 155 amino acids. To simplify the 1H NMR spectra, two fully deuterated enzymes bearing several protonated amino acids were prepared from an RNase HI overproducing strain of E. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, Ile, Val, and Leu. The other was labeled by only protonated His and Ile. The 2D 1H NMR spectra of these deuterated RNase HI proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.
 ...
PMID:1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids. 133 Jan 30

The capacity of some Escherichia coli (E. coli) ribosomal proteins to bind to tRNA and to hydrolyse their aminoacylated derivatives has been analysed. The following results were obtained: (1) The basic proteins L2, L16 and L33 and S20 bound f[3H]Met-tRNA to a similar extent as the total proteins from 30 S (TP30) or 50 S (TP50) when tested by nitrocellulose filtration, in contrast to the more acidic proteins L7/L12 and S8. (2) The proteins of the peptidyltransferase centre, L2 and L16, showed no distinct specificity, binding various charged tRNAs from E. coli and Saccharomyces cerevisiae (S. cerevisiae). (3) A number of isolated ribosomal proteins hydrolysed aminoacyl-tRNA as assessed by trichloroacetic acid precipitation, in contrast to the TP30 and TP50. (4) The loss of radiolabel from Ac[14C]Phe-tRNA and from [14C]tRNA in the presence of these proteins could not be prevented by RNasin, a ribonuclease inhibitor, whereas that mediated by a sample of non-RNase-free bovine serum albumin was inhibited. (5) When double-labelled, Ac[3H]Phe-[14C]tRNA was incubated with L2 both radiolabels were lost, indicating that this potential candidate for a peptidyltransferase enzyme does not specifically cleave the ester bond between the aminoacyl residue and the tRNA.
 ...
PMID:The complex between ribosomal proteins and aminoacyl-tRNA: the interactions and hydrolytic activities are not confined to the proteins L2 and L16 of Escherichia coli ribosomes. 218 27

The gene for ribonuclease T1 from Aspergillus oryzae has been chemically synthesized using the segmental support technique. An Escherichia coli clone producing the ribonuclease at high levels was constructed by linking the gene downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E. coli), using the secretion cloning vector pIN-III-ompA2. This strategy was employed in order to circumvent a possible toxic effect of the gene product on the host cell. Active ribonuclease containing four additional amino acids at the N-terminus could be isolated from the periplasmic fraction of the host. The final yield after purification was 20 mg enzyme/l liquid culture. With respect to immunological, catalytic and specific behaviour, no qualitative differences could be detected between the enzyme from the over-producing E. coli strain and ribonuclease T1 isolated from A. oryzae.
 ...
PMID:Expression of the chemically synthesized gene for ribonuclease T1 in Escherichia coli using a secretion cloning vector. 313 Nov 42

In this study, the efficacy of an anti-ras ribozyme in reversing a transformed phenotype was investigated. A murine NIH/3T3-derived cell line, designated 2-12, contains an inducible Ha-ras oncogene, which is regulated by the Escherichia coli (E. coli) lac operator/repressor system, and displays a transformed phenotype after isopropyl-beta-D-thiogalactoside induction. To reverse the transformed characteristics, the ribozyme, which specifically targets the Ha-ras oncogene at the codon 12 mutation site (GGC to GUC), was transfected into 2-12 cells. Two (ribZ4 and ribZ7) clones were subsequently selected and analyzed for their transforming features. Our results show that, in the transfectants, ribozyme gene expression was detected, and the target Ha-ras transgene was expressed at basal levels. Their phenotypic responses, including morphology, cell growth rate, colony-formation efficiency and tumorigenicity in mice with severe combined immunodeficiency were more similar to those of NIH/3T3 than 2-12 transformed cells. Directly injecting the ribozyme DNA into tumors induced by transformed 2-12 cells in BALB/c mice also caused tumor regression. The enzymatic cleavage products of the ribozyme acting on mutant Ha-ras mRNA in vivo were detected by primer-extension analysis. These results indicate that the ribozyme were designed exhibits a site-specific ribonuclease function that effectively abrogates Ha-ras-oncogene-induced transformation, and this unique anti-Ha-ras property should shed light on the development of strategies against the Ha-ras-oncogene-initiated malignancy.
 ...
PMID:A ribozyme specifically suppresses transformation and tumorigenicity of Ha-ras-oncogene-transformed NIH/3T3 cell lines. 903 Feb 47

Bovine seminal ribonuclease (BSRNase) is an unusual member of the ribonuclease superfamily, because of its remarkable anti-tumour and immunosuppressive properties. We describe here the construction, expression, purification and characterization of a panel of six immunotoxins based upon this enzyme and show that we can increase its anti-tumour activity by over 2 x 10(4)-fold. This is achieved by improving tumour cell targeting using a single-chain Fv (scFv) directed against the oncofetal antigen placental alkaline phosphatase. As well as the simple scFv-BSRNase fusion protein, we have constructed five other derivatives with additional peptides designed to improve folding and intracellular trafficking and delivery. We find that the molecule most cytotoxic to antigen (PLAP)-positive cells in vitro is one that contains a C-terminal 'KDEL' endoplasmic reticulum retention signal and a peptide sequence derived from diphtheria toxin. All these molecules are produced in Escherichia coli (E. coli) as insoluble inclusion bodies and require extensive in vitro processing to recover antigen binding and ribonuclease activity. Despite incomplete ribonuclease activity and quaternary assembly, these molecules are promising reagents for specific chemotherapy of cancer and are potentially less harmful and immunogenic than current immunotoxins.
 ...
PMID:Design, characterization and anti-tumour cytotoxicity of a panel of recombinant, mammalian ribonuclease-based immunotoxins. 948 8

The solution structure of ribonuclease HI (RNase HI) from Escherichia coli (E. coli), a protein of 155 residues, was determined. Three-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) was used to obtain 1,424 distance constraints between individually assigned polypeptide chain hydrogen atoms. Supplemental geometric constraints of 90phi angles and 12chi1 angles, and the distance constraints of 66 hydrogen bonds were experimentally derived. Using the DADAS90 program that calculates structures in dihedral angle space, 15 structures satisfying almost all constraints were obtained. The average root mean square deviation (RMSD) from the mean structure was 0.75 A for backbone atoms. The RMSD for backbone atoms between the representative NMR structure with the smallest constraint violation and crystal structures was within 1.2 A. Although the NMR and crystal structures thus resemble one another, a significant discrepancy was observed in a region termed 'basic protrusion.' The discrepancy observed in NMR experiments is explained by fluctuation in this region.
 ...
PMID:NMR structure of ribonuclease HI from Escherichia coli. 1104 Dec 41

The Escherichia coli (E. coli) ribonuclease E protein (RNase E) is implicated in the degradation and processing of a large fraction of RNAs in the cell. To understand RNase E function in greater detail, we developed an efficient selection method for identifying nonfunctional RNase E mutants. A subset of the mutants was found to display a dominant-negative phenotype, interfering with wild-type RNase E function. Unexpectedly, each of these mutants contained a large truncation within the carboxy terminus of RNase E. In contrast, no point mutants that conferred a dominant-negative phenotype were found. We show that a representative dominant-negative mutant can form mixed multimers with RNase E and propose a model to explain how these mutants can block wild-type RNase E function in vivo.
 ...
PMID:Identification and analysis of Escherichia coli ribonuclease E dominant-negative mutants. 1620 12

Many macromolecules in the cell function by forming multi-component assemblies. We have applied the technique of small angle neutron scattering to study a nucleic acid-protein complex and a multi-protein complex. The results illustrate the versatility and applicability of the method to study macromolecular assemblies. The neutron scattering experiments, complementing X-ray solution scattering data, reveal that the conserved catalytic domain of RNase E, an essential ribonuclease in Escherichia coli (E. coli), undergoes a marked conformational change upon binding a 5'monophosphate-RNA substrate analogue. This provides the first evidence in support of an allosteric mechanism that brings about RNA substrate cleavage. Neutron contrast variation of the multi-protein TIM10 complex, a mitochondrial chaperone assembly comprising the subunits Tim9 and Tim10, has been used to determine a low-resolution shape reconstruction of the complex, highlighting the integral subunit organization. It shows characteristic features involving protrusions that could be assigned to the six subunits forming the complex.
 ...
PMID:Complementing structural information of modular proteins with small angle neutron scattering and contrast variation. 1827 Jun 93

Waterborne pathogens usually pose a global threat to animals and human beings. There has been a growing demand for convenient and sensitive tools to detect the potential emerging pathogens in water. In this study, a lab-on-a-chip (LOC) device based on the real-time immuno-NASBA (immuno-nucleic acid sequence-based amplification) assay was designed, fabricated and verified. The disposable immuno-NASBA chip is modelled on a 96-well ELISA microplate, which contains 43 reaction chambers inside the bionic channel networks. All valves are designed outside the chip and are reusable. The sample and reagent solutions were pushed into each chamber in turn, which was controlled by the valve system. Notably, the immuno-NASBA chip is completely compatible with common microplate readers in a biological laboratory, and can distinguish multiple waterborne pathogens in water samples quantitatively and simultaneously. The performance of the LOC device was demonstrated by detecting the presence of a synthetic peptide, ACTH (adrenocorticotropic hormone) and two common waterborne pathogens, Escherichia coli (E. coli) and rotavirus, in artificial samples. The results indicated that the LOC device has the potential to quantify traces of waterborne pathogens at femtomolar levels with high specificity, although the detection process was still subject to some factors, such as ribonuclease (RNase) contamination and non-specific adsorption. As an ultra-sensitive tool to quantify waterborne pathogens, the LOC device can be used to monitor water quality in the drinking water system. Furthermore, a series of compatible high-throughput LOC devices for monitoring waterborne pathogens could be derived from this prototype with the same design idea, which may render the complicated immuno-NASBA assays convenient to common users without special training.
 ...
PMID:Compatible immuno-NASBA LOC device for quantitative detection of waterborne pathogens: design and validation. 2214 18