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

Abstract

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...

Structural and functional studies of the Thermus thermophilus 16S rRNA methyltransferase RsmG

“…1E). Methylation of the N7 of guanosine residues in RNA imparts on the glycosidic bond susceptibility to cleavage with aniline after reduction of the N7-C8 bond with NaBH 4 (Peattie 1979;Zueva et al 1985). This cleavage is detectable by primer extension with reverse transcriptase (RT).…”

“…To induce strand scission at m 7 G residues, rRNA was treated with NaBH 4 and aniline (Krol and Carbon 1989); reactions included dimethyl sulfate-methylated carrier tRNA as described (Zueva et al 1985). Modified RNA was subjected to primer extension using AMV reverse transcriptase (Seikagaku) and a 32 P-end-labeled oligonucleotide primer, Tth 16S-rsmG1, complementary to positions 565 to 548 of T. thermophilus 16S rRNA.…”

The aminoglycoside resistance methyltransferase Sgm impedes RsmF methylation at an adjacent rRNA nucleotide in the ribosomal A site

“…Sites of m 7 G methylation in the rRNAs were reduced with sodium borohydride (NaBH 4 ) and cleaved via aniline-induced b-elimination (Peattie 1979). This reaction is generally incomplete (Douthwaite et al 1983) but was improved by inclusion of hypermethylated tRNA carrier in the reactions (Zueva et al 1985). A 59-32 P-end-labeled deoxynucleotide primer, complementary to 16S rRNA nucleotides 1459-1479, was hybridized to the rRNA and extended with AMV reverse transcriptase (Finnzymes) (Stern et al 1988).…”

Identification of the RsmG Methyltransferase Target as 16S rRNA Nucleotide G527 and Characterization of Bacillus subtilis rsmG Mutants

“…The RNAs were cleaved at N7-methylguanosine positions by reduction with NaBH 4 , followed by ␤-elimination with acetic acid-aniline (26,33); a tRNA carrier, hypermodified at N7 of guanosines by dimethyl sulfate treatment, was added to enhance cleavage at the N7-methylated guanosines in the rRNA (34). The rRNAs were scanned using a series of primers by reverse transcriptase extension (32).…”