The Primary Structure of tRNAIIArg from Brewers' Yeast. 2. Partial Digestion with Ribonuclease T1 and Derivation of the Complete Sequence

Abstract

Several large oligonucleotides obtained by partial digestion of tRNAFg from brewers' yeast with ribonuclease T, have been isolated and their sequences have been established. These results and those described in the preceding paper permitted the derivation of the complete primary structure of this tRNA.Larger fragments than those obtained after complete pancreatic and T, ribonucleases digestions of tRNAEg had to be obtained in order to derive the complete sequence of this tRNA. These large fragments have been produced by partial digestion with T, ribonuclease. This paper reports the conditions of controlled cleavage of tRNA;\;p with RNase TI, the separations and the analyses of the fragments obtained and the derivation of the complete primary structure of this tRNA. MATERIALS AND METHODS General MethodsSource of tRNA? and of the various enzymes, thin-layer chromatography and high-voltage electrophoresis conditions, methods of analysis and of identification of oligonucleotides and other general techniques have been described elsewhere [l -31. Polyacrylamide Gel ElectrophoresisThe action of T, ribonuclease was followed by polyacrylamide gel electrophoresis of the digests. The gels were scanned at 254 nm using a Vernon photoAbbreviations for polynucleotides follow the recommendations of IUPAC-IUB Commission on Biochemical Nomenclature for abbreviations and symbols for nucleic acids, polynucleotides and their constituents see Eur. J. Biochem. 15, 203 (1970).Definirion. A260 unit, amount of material in 1 ml of solution which gives an absorbance of 1 at 260 nm in a I-cm light path.Enzymes. Ribonuclease TI (EC 3.1.4.8); alkaline phosphatase (EC 3.1.3.1).densitometer. The optimal partial hydrolysis conditions concerning enzyme and magnesium concentrations and digesting time were determinated from the electrophoretic patterns.Partial Digestion of tRNA? with Ribonuclease T, 300 A260 units of purified tRNAEg were incubated with 200 units of RNase T1 in 12 ml of a 0.05 M Tris-HC1 buffer (pH 7.5) 0.02 M Mg C12 for 30 min at 0 "C.Enzyme was then removed by treating twice with an equal volume of phenol saturated at 0 "C with 0.05 M Tris-HC1 buffer (pH 7.5) containing 0.02 M Mg C12. The phenol phases were pooled and backextracted twice with half a volume of water. The pooled aqueous fractions were washed four times with two volumes of ethyl ether. This solution, brought to a 7 M urea concentration by addition of solid urea, was loaded on a DEAE-cellulose 7 M urea column. Separation of Large OligonucleotidesThe T, partial digest was first submitted to DEAEcellulose-7 M urea-Tris-HC1 0.02 M (pH 7.3) column chromatography (Fig. 1). Each peak of oligonucleotides isolated from the DEAE-cellulose column at pH 7.3 was diluted five-fold with 7 M urea and loaded on an other DEAE-cellulose column. This column was then equilibrated with 7 M urea-HC1, pH 3, and eluted by a linear sodium chloride gradient. Urea and salt were removed from the pooled oligonucleotide fractions as previously described [l -31.Eur. J. Biochem. 56 (1975)

Detection of Inosine on Transfer RNAs without a Reverse Transcription Reaction

“…In recent years, conflicting evidence has emerged regarding the I34 modification status of tRNA Arg ACG in higher eukaryotes . While I34 could be readily detected by sequencing of tRNA Arg ACG in Escherichia coli , Mycoplasma capricolum , and Saccharomyces cerevisiae , it could not be detected in nematodes, mosses, higher plants, insects, or mammals. − However, the presence of I34 on human tRNA Arg ACG has been documented by other means, suggesting that at least in some of these cases RT-based methods may be leading to artifactual results.…”

Arginine aminoacylation identity is context-dependent and ensured by alternate recognition sets in the anticodon loop of accepting tRNA transcripts.

“…How is arginine identity expressed in yeast? Yeast cells bear three cytosolic tRNAArg isoacceptors (Weissenbach et al, 1975;Keith and Dirheimer, 1980). The sequences of species II, Illa and IlIb are displayed in Figure LA; that of species I is not yet known.…”

A previously unidentified activity of yeast and mouse RNA:pseudouridine synthases 1 (Pus1p) on tRNAs

“…The mPus1p activity was tested in Dpus1 cells and compared with in vitro results. Modification of both tRNAs and U2 snRNA was tested and we investigated whether scPus1p and mPus1p exhibit one or both of the yet unidentified yeast tRNA:C-synthase activities, namely, pseudouridylation at position 1 in the cytoplasmic tRNA Arg (ACG) and tRNA Lys (UUU) (Madison and Boguslawski 1974;Weissenbach et al 1975) and pseudouridylation FIGURE 1. Sequences and secondary structures of the S. cerevisiae tRNA substrates used in this work.…”