An enzymatic approach for localization of oligodeoxyribonucleotide binding sites on RNA

Mankin, Alexander S.; Skripkin, Eugene; Chichkova, Nina V.; Копылов, А. М.; Bogdanov, Alexei A.

doi:10.1016/0014-5793(81)80378-x

Cited by 34 publications

(16 citation statements)

References 8 publications

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“…These observations are confirmatory of many earlier findings concerning the surface position of the 3' RNA terminus, its interaction with extraribosomal components of the protein-synthesizing apparatus, and the nature of the oligonucleotides detached after nuclease digestion (6,(36)(37)(38)(39)(40)(41)(42)(43)(44)(45); and the location by immuno-electron microscopy of the 3' end on or near the "collar" (46-49). Korn's observations add to the prevailing picture.…”

Section: Discussionsupporting

confidence: 89%

Long range RNA-RNA interactions in the 30 S ribosomal subunit ofE. coli

et al. 1985

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We have attempted to identify long-range interactions in the tertiary structure of RNA in the E. coli 30 S ribosome. Native subunits were cleaved with ribonuclease and separated into nucleoprotein fragments which were deproteinized and fractionated into multi-oligonucleotide complexes under conditions intended to preserve RNA-RNA interactions. The final products were denatured by urea and heat and their constituent oligonucleotides resolved and sequenced. Many complexes contained complementary sequences known to be bound together in the RNA secondary structure, attesting to the validity of the technique. Other co-migrating oligonucleotides, not joined in the secondary structure, contained mutually complementary sequences in locations that allow base-pairing interaction without disrupting pre-existing secondary structure. In seven instances the complementary relationship was found to have been preserved during phylogenetic diversification.

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Section: Discussionsupporting

confidence: 89%

Long range RNA-RNA interactions in the 30 S ribosomal subunit ofE. coli

et al. 1985

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“…RNase H cleavage in the presence of synthetic oligodeoxyribonucleotide d(C-C-C-A-G-T-A-A-T) was effected as described earlier [4].…”

Section: Cleavage Of 16s Rrna With Rnase Hmentioning

confidence: 99%

“…It is based on a site-specific cleavage of 16s rRNA with RNase H in the presence of oligodeoxyribonucleotide, complementary to a definite segment of the rRNA [4]. The nonanucleotide d(C-C-C-A-G-T-A-A-T), which is complementary to the region 560 -568 of the 16s rRNA, was used.…”

Section: Localization Of the 16s Rrna Cleavage Pointmentioning

confidence: 99%

Specific fragmentation of tRNA and rRNA at a 7-methylguanine residue in the presence of methylated carrier RNA

et al. 1985

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1. The reaction of site-specific cleavage of tRNA at a 7-methylguanine residue, including subsequent treatment with sodium borohydride and aniline [Wintermeyer, W. and Zachau, H. G. (1975) FEBS Lett. 58,306-3091, was shown to work only within a certain range of tRNA concentrations (higher than 30 pM). The Escherichia coli 16s rRNA, which contained a unique m7G (position 527), could not be split by this method when taken at any concentration.2. It was found that the presence of statistically methylated carrier RNA in the reaction mixture at the borohydride stage significantly stimulates site-specific fragmentation of 16s rRNA and 32P-labeled tRNAs.3. Direct sequencing proved that 16s rRNA and tRNAs are cleaved by this procedure successfully at the m7G residue.4. The E. coli 16s rRNA was preparatively cleaved by the described procedure into two fragments. The 5'-terminal fragment (1 -526) and the 3'-terminal fragment (528 -1542) were isolated in the pure form and their secondary structure investigated by the circular dichroism method. The results of this study showed that the secondary and tertiary structures of the 5'-terminal one-third of the 16s rRNA are at least as ordered as those of intact 16s rRNA or its 3'4erminal two-thirds.The site-specific fragmentation of a polynucleotide chain is widely used for the study of the structure and functions of RNA and ribonucleoprotein complexes. Apart from the enzymatic approaches El-41, the chemical methods of splitting native RNAs at modified bases hold promise. Previously Zachau and co-workers described the procedure for splitting the yeast tRNAPhe at some minor nucleotides [5-71. The procedure of tRNA cleavage at a 7-methylguanine residue was based on reducing this base with sodium borohydride and subsequently splitting the polynucleotide chain at the reduced base with aniline.However, we have not been successful in our attempts to make direct use of this method for splitting the Escherichiu coli 16s rRNA at the unique 7-methylguanine residue or the 3'-terminally labeled yeast [32P]tRNAPhe. A closer study of the reaction has shown that the addition of the methylated RNA-carrier (methyl-RNA) at the borohydride reduction stage considerably enhances the efficiency of the reaction and makes it possible to cleave site-specifically 16s rRNA or tRNA with a 50-70% yield. Using this method we could cleave relatively large amounts of E. coli 16s rRNA at the unique residue of 7-methylguanine at position 527. A 526-nucleotide-long 5'-terminal fragment and a 1 01 5-nucleotidelong 3'-terminal fragment have been isolated and their secondary structure investigated by the circular dichroism (CD) method. MATERIALS AND METHODSThe 16s rRNA was isolated by phenol extraction from the 30s ribosomal subunits of Escherichia coli (strain MRE600).Abbreviations. Methyl-RNA, carrier RNA statistically methylated with dimethylsulfate; CD, circular dichroism.The phenylalanine and valine tRNA from yeast were kindly provided by Dr T.V. Venkstern. The RNase H preparation from E. coli was furnished by...

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“…In some cases the venom digestion was followed by a further digestion with ribonuclease H in the presence of the hexanucleotide d-(CTTCCC) (cf. 11,12,4). Cross-linked RNA complexes were isolated from these digests by two-dimensional gel electrophoresis and subjected to oligonucleotide analysis by our established methodology (3,4), and we were able to identify a total of four tertiary structural and two secondary structural cross-links.…”

Section: Introductionmentioning

confidence: 99%

Investigation of the tertiary folding ofEscherichia coli16s RNA byin situintra-RNA crosslinking within 30S ribosomal subunits

Atmadja

Brimacombe

Blöcker³

et al. 1985

Nucl Acids Res

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Intra-RNA cross-links were introduced into E. coli 30S ribosomal subunits by mild ultraviolet irradiation. The subunits were partially digested with cobra venom nuclease, followed in some cases by a second partial digestion with ribonuclease H in the presence of the hexanucleotide d-(CTTCCC). The cross-linked RNA complexes were separated by two-dimensional gel electrophoresis and the sites of cross-linking analysed by our published procedures. Tertiary structural cross-links in the 16S RNA were identified between positions 31 and 48, between oligonucleotides 1090-1094 and 1161-1164, and between oligonucleotides 1125-1127 and 1280-1281. The first of these imposes a rigid constraint on the relative orientations of helices 3 and 4 of the 16S secondary structure. A further tertiary cross-link (which could not be precisely localised) was found between regions 1-72 and 1020-1095, and secondary structural cross-links were identified between positions 497 and 545-548, and positions 1238-1240 and 1298.

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An enzymatic approach for localization of oligodeoxyribonucleotide binding sites on RNA

Cited by 34 publications

References 8 publications

Long range RNA-RNA interactions in the 30 S ribosomal subunit ofE. coli

Long range RNA-RNA interactions in the 30 S ribosomal subunit ofE. coli

Specific fragmentation of tRNA and rRNA at a 7-methylguanine residue in the presence of methylated carrier RNA

Investigation of the tertiary folding ofEscherichia coli16s RNA byin situintra-RNA crosslinking within 30S ribosomal subunits

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