The nucleotide sequence of two homogeneic Drosophila melanogaster tRNATyr isoacceptors: application of a rapid tRNA anticodon sequencing method using S-1 nuclease

Abstract

The nucleotide sequence of the two major Drosophila melanogaster tRNATyr isoacceptors was determined to be pC-C-U-U-C-G-A-U-A-m2G-C-U-C-A-G-D-D-G-G-acp3 U-A-G-A-G-C-m2(2)G-G-psi-G-G-A-C-U-G/Q-psi-A-m1G-A-Um-C-C-A-U-A-G-m7 G-D-C-G-C-U-G-G-U(T)-psi-C-A-m1A-A-U-C-C-G-G-C-U-C-G-A-A-G-G-A-A-C-C-AOH . The two isoacceptors differ by the presence of a G or a Q in the wobble position. Both contain a partial modification in position 54 (U/T). Thus, these tRNAs are transcribed from a single gene (or many genes with identical sequences). A fast and sensitive postlabeling method for sequencing tRNA anticodons is described. Nuclease S-1-treated tRNA is labeled with 5[32P]-pCp using T-4 RNA ligase. The tRNA fragments are then separated on 7 M urea/20% PAA gels. After autoradiography the RNA is eluted and digested with T-2 RNase. The nature of the labeled nucleotides is determined by two-dimensional thin-layer chromatography. The same method can be used to determine the 5' sequence of a tRNA by 3' labeling 5' tRNA halves with 5[32P]-pCp and subsequent chemical sequencing.