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:3.1.30.1 (S1 nuclease)
3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this paper we report on the thermal unfolding of the tRNA-like structure present at the 3' end of turnip yellow mosaic virus (TYMV) RNA. Diethyl pyrocarbonate (DEP), sodium bisulphite, nuclease S1 and ribonuclease T1 were used as structure probes at a broad range of temperatures. In this way most of the nucleotides present in the tRNA-like moiety were analysed. The melting behaviour of both secondary and tertiary interactions could be followed on the basis of the temperature dependent accessibility of the individual nucleotides or bases towards the various probes. The three-dimensional model of the tRNA-like domain (Dumas et al., J. Biomol. Struct. and Dyn. 4, 707 (1987] was supported by the results to a large extent. The interactions occurring between the T- and D-loop appear to be more complex than proposed in the latter model. Additional evidence for the presence of the RNA pseudoknot (Rietveld et al., Nucleic Acids Res. 10, 1929 (1982] was derived from the fact that the three coaxially stacked helical segments in the aminoacylacceptor arm displayed different melting transitions under certain experimental conditions. Aspects of melting behaviour and thermal stability of double helical regions within the tRNA-like structure are discussed, as well as the applicability of nucleases and modifying reagents at various temperatures in the analysis of RNA structure.
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PMID:Temperature dependent chemical and enzymatic probing of the tRNA-like structure of TYMV RNA. 283 23

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

Accessible sites in the 5' noncoding region of the rabbit alpha- and beta-globin mRNAs were identified and compared in deproteinized RNA and in the mRNAs engaged in translation in the reticulocyte lysate. Preparations of RNA and lysate were subjected to limited nuclease digestion by RNase T1 and Neurospora endonuclease, and the cleavage sites were analyzed by a nuclease S1 mapping procedure. The free alpha-globin mRNA contained few nuclease-sensitive sites and its initiation codon AUG was masked. The free beta-globin mRNA contained a larger number of accessible sites and its AUG was highly exposed. The distribution of sensitive sites differed considerably in the lysate. In both mRNA species, a site near the 5' terminus became the one most accessible to Neurospora endonuclease. Also the accessibility of the AUG in beta-globin mRNA decreased considerably. The distribution of accessible sites in the lysate was the same when the mRNAs were undergoing rapid initiation and when initiation became limited after prolonged incubation. Inhibition of initiation by the cap analogue 7-methylguanosine 5'-triphosphate was accompanied by increased sensitivity of some of the sites in both mRNA species. One of the accessible sites in each mRNA species had a sequence complementary to the 3'-terminal portion of the 18S ribosomal RNA.
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PMID:Structural features of the 5' noncoding region of the rabbit globin messenger RNAs engaged in translation. 300 32

The ribosomal 5S RNA gene from the rrnB operon of E. coli was mutagenised in vitro using a synthetic oligonucleotide hybridised to M13 ssDNA containing that gene. The oligonucleotide corresponded to the 5S RNA sequence positions 34 to 51 and changed the guanosine at position 41 to a cytidine. The DNA containing the desired mutation was identified by dot blot hybridisation and introduced back into the plasmid pKK 3535 which contains the total rrnB operon in pBR 322. Plasmid coded 5S rRNA was selectively labeled with 32p using a modified maxi-cell system, and the replacement of guanosine G41 by cytidine was confirmed by RNA sequencing. The growth of cells containing mutant 5S rRNA was not altered by the base change, and the 5S rRNA was processed and incorporated into 50S ribosomal subunits and 70S ribosomes. The structure of wildtype and mutant 5S rRNA was compared by chemical modification of accessible guanosines with kethoxal and limited enzymatic digestion using RNase T1 and nuclease S1. These results showed that the wildtype and mutant 5S rRNA do not differ significantly in their structure. Furthermore, the formation, interconversion and stability of the two 5S rRNA A- and B-conformers are unchanged.
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PMID:Oligonucleotide directed mutagenesis of Escherichia coli 5S ribosomal RNA: construction of mutant and structural analysis. 609 Oct 46

Masked and exposed sites in rabbit beta-globin messenger RNA were identified through S1 nuclease mapping of RNase T1 cleavage sites. Sites exposed to this enzyme were compared in deproteinized polysomal RNA and in mRNA in its native configuration in reticulocyte extracts. The analysis showed that most of the 3' non-coding region is well accessible to the enzyme, both in deproteinized RNA and in the cell extract. A possible protecting function for the poly(A) sequence is suggested by the fact that molecules with very short poly(A) segments were cleaved preferentially in this region. The G residues in the 5' non-coding region were inaccessible to RNase T1. A highly sensitive site adjacent to the initiation AUG codon was evident in the deproteinized RNA. This site was far less accessible to the enzyme in the mRNA associated with ribosomes in the cell extract. The first 150 nucleotides in the coding region showed very little susceptibility to digestion by the enzyme, in deproteinized RNA as well as in the cell extracts. Preparations of untreated mRNA showed the occurrence of truncated molecules, apparently generated by cleavage by endogenous nucleases. These cleavages were most prevalent in the two non-coding regions. They occurred at sites containing A-U sequences in the 3' non-coding region, and at sites with different sequences in the 5' non-coding region. Incubation of cell extracts at 37 degrees C did not cause any increase in these endogenous cleavages. It is suggested that they may have been generated in the intact cells, possibly as part of the mRNA degradation process in maturing reticulocytes.
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PMID:Configuration of beta-globin messenger RNA in rabbit reticulocytes. Identification of sites exposed to endogenous and exogenous nucleases. 609 46

The major 5'-termini of human adenovirus type 2 early gene block 4 mRNA were sequenced. Poly(A+) polyribosomal RNA was isolated from Ad2 early infected cells, the 5'-terminal m7GPPP removed and the 5'-OH of the penultimate 2'-0-methylated nucleotide labeled with [gamma-32P]ATP using polynucleotide kinase. Ad2 E4 mRNA was purified by hybridization to the Ad2 EcoRI-C fragment and was digested with RNase T1. The resulting oligonucleotides were resolved by two dimensional paper electrophoresis-homochromatography. Four major and 3-4 minor 5'-terminal sequences were identified and characterized. The sequence of the 5'-terminal structures of the major four termini are: (1) m7GpppUmU(m)UUACACUGp, (2) m7GpppUmU(m)UACACUGp, (3) m7GpppUmU(m)ACACUGp, and (4) m7Gppp(m6)AmC(m)ACUGp. These major 5'-terminal sequences were aligned with nucleotide 325, 326, 327, and 329 from the righthand end of the known Ad2 DNA sequence (1) in the region mapped as the 5'-terminus of E4 mRNA by electron microscopy (2,3) and S1 nuclease-gel (4) mapping. Two potential ribosomal binding sites and an initiator codon were found at 40 to 65 nucleotides and about 80 nucleotides, respectively, from these heterogenous 5'-termini. Ad2 E4 major mRNA species appear to be unique since mRNA molecules initiate at a pyrimidine, perhaps by RNA polymerase stuttering, or they are products of an unusual type of RNA processing.
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PMID:Nucleotide sequences and mapping of novel heterogenous 5'-termini of adenovirus 2 early region 4 mRNA. 616 92

Sat-RNA is one of several replicating satellite RNAs which have been isolated from RNA encapsidated in cucumber mosaic virus (CMV) and which are totally dependent on CMV for replication. The 336 residue sequence of Sat-RNA obtained using the dideoxynucleotide chain termination and partial enzymic digestion procedures shows only a few short stretches (up to 11 residues) of sequence homology with one of the three CMV genomal RNAs so far sequenced. Sat-RNA has 88% sequence homology with another, previously sequenced, satellite RNA of CMV, CARNA 5. Analysis of partial digests of 5'- or 3' -32P-Sat-RNA with nuclease S1 or RNase T1 under non-denaturing conditions showed that only about 10% of the residues in Sat-RNA were cleaved. Further data on base-paired segments of Sat-RNA were obtained using digestion with RNase T1 followed by electrophoretic fractionation of the resulting fragments under both non-denaturing and denaturing conditions. On the basis of this data, a complete secondary structure model is proposed for Sat-RNA with 52% of its residues involved in base pairs. A prominent hairpin at the 3'-terminus of Sat-RNA shows considerable sequence and structural homology with parts of the 3'-terminal tRNA-like structure of the CMV genomal RNAs.
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PMID:Satellite RNA of cucumber mosaic virus forms a secondary structure with partial 3'-terminal homology to genomal RNAs. 618 89

The structures of the two stable conformers of Escherichia coli 5 S RNA, the and B form, were compared. Information about the structures were obtained using the methods of limited enzymatic hydrolysis and chemical modification of accessible nucleotides. Base-specific modifications were performed for adenosines and cytidines using diethylpyrocarbonate and dimethylsulfate in combination with a strand-scission reaction at the modified site. Base-specific (RNase T1) as well as conformation-specific (nuclease S1, cobra venom nuclease) enzymes were employed for the limited enzymatic hydrolysis. Clear differences in the accessibility of the two 5 S RNA conformers to the enzymes and the chemical reagents were established and the regions with altered reactivities were localized in the 5 S RNA structure. The results are consistent with the disruption of the secondary structural interactions in helix II and partly in helices III and IV during the transition from the A to the B form. (The numbering of the helices is according to the generally accepted Fox and Woese model.) In addition some regions presumably involved in the tertiary structure are distorted. There is evidence, however, for the new formation of structural regions between two distant sites in the 5 S RNA B form. The results enable us to refine the existing 5 S RNA A-form model and provide insight into the structural dynamics that lead to the formation of the 5 S RNA B form.
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PMID:Escherichia coli 5S RNA A and B conformers. Characterisation by enzymatic and chemical methods. 620 22

Poly(7-deazaguanylic acid) was enzymatically synthesized by the polymerization of 7-deazaguanosine 5'-diphosphate with polynucleotide phosphorylase from Micrococcus luteus in high yield. The homopolymer showed a similar thermal and total hypochromicity to poly(G) at the long wavelength absorption maximum. No sigmoid melting profile was observed for poly(c7G) as is found for poly(G), implying a single-stranded structure in aqueous solution. From the circular dichroism spectra it can be concluded that the 7-deazapurine nucleotide is much more flexible than the purine nucleotide. In analogy to poly(G), the homopolymer poly(c7G) forms a 1:1 complex with poly(C) under neutral conditions, melting at a similar temperature to the poly(G) complex. However, at pH 2.5, where a poly(G) X 2poly(C) complex is observed, poly(c7G) still binds only one poly(C) strand. This is due to the lack of N-7 in poly(c7G), not allowing Hoogsteen base pair formation, which occurs with poly(G). RNase T1 cleaves poly(c7G), indicating that N-7 of guanosine is not a requirement for nucleotide binding to the enzyme, as has been suggested. Because of the single-stranded structure of poly(c7G), the polynucleotide chain is rapidly hydrolyzed by the single-strand-specific nuclease S1, whereas multistranded poly(G) is completely resistant.
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PMID:Poly(7-deazaguanylic acid), the homopolynucleotide of the parent nucleoside of queuosine. 628 79

Escherichia coli translational initiation factor 3 (IF3) may be crosslinked to the 3' end of 16S RNA in 30S ribosomal subunits. In order to determine the sequence to which IF3 may bind in vivo, samples of 5'-32P labelled 3' terminal 49-nucleotide fragment of 16S RNA were incubated 5 min. at 37 degrees in 40 mM Tris-HOAc, pH 7.4, 100 mM NaCl, 1 mM Mg (OAc)2, 1 mM ZnSO4, with or without IF3, then reacted a further 5 min with nuclease S1, RNase T1, or RNase A. Base pairing between the 5' and 3' legs of the fragment occurs in the absence of IF3, but is disrupted by IF3 binding. IF3 appears to protect some residues near the 5' end of the fragment (U1495, A1499, A1500, A1502, and A1503) from nuclease S1, and potentiates S1 attack on others (G1494, G1497, C1501, G1504, G1505, U1506, G1517, G1529, G1530, and C1533). A series of equimolar reactions at increasing dilution imply an association constant range of 1.4-7.0 X 10(7) M-1.
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PMID:Nuclease mapping of the secondary structure of the 49-nucleotide 3' terminal cloacin fragment of Escherichia coli 16s RNA and its interactions with initiation factor 3. 634 66


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