Yeast tRNA^Leu_UAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods

Abstract

A second major species of leucine tRNA, t R N A e G (formerly designated t R N A e A ) was purified from baker's yeast in a three-step procedure entailing BD-cellulose chromatography in the presence and absence of Mg2+ and Sephadex G-100 gel filtration. Results of aminoacylation and partial RNase TI digestion experiments showed that this tRNA retains a native conformation under conditions that denature yeast tRNAscAA (tRNAP").The primary structure of baker's yeast tRNA?lG was elucidated by application of sensitive radioactive isotope derivative ('postlabeling') methods. Complete RNaseTl and A and partial RNase Uz fragments, prepared from non-radioactive tRNA and 5'-half and 3'-half molecules, were separated by two-dimensional polyethyleneimine-cellulose anion-exchange thin-layer chromatography and isolated by a novel micropreparative procedure affording high yields of these compounds in sufficient purity for subsequent tritium derivative analysis. Base composition and sequence of oligonucleotides were analyzed by tritium derivative methods. Molar ratios of the fragments were determined from the radioactivity of 3H-labeled nucleoside trialcohols in combination with base analysis. 2I-O-Methylated guanosine was characterized using the [y-32P]ATP/polynucleotide kinase reaction. The analysis of classical complete and partial RNase digests by the tritium derivative methods yielded the complete nucleotide sequence of the RNA. A total of about 20 A260 units of the RNA was used for analysis, i.e. considerably less material than required for conventional spectrophotometric analysis.A different sequencing approach, consisting of a combination of 'readout sequencing' with tritium sequencing of complete RNase TI and A fragments, was applied to the 3'-half molecule. The 3'-half molecule was labeled with 32P at its 5' terminus, partially degraded with RNases TI, UZ, and Phy, and with alkali, and subjected to polyacrylamide gel electrophoresis. The sequence was read off the gel on the basis of cleavage patterns and size of the fragments. While the readout procedure provided only the positions of A, U, C, and G residues in the chain, additional information from tritium derivative analysis was utilized to define the positions of the modified nucleosides. The readout sequencing procedure was found to require less than 0.01 unit of RNA and the analysis of the complete fragments about 6 A260 units.Interesting structural features of tRNAfLG are (a) the location of unique, leucine tRNA isoacceptor-specific sequences next to U-8, a constant nucleotide participating in synthetase recognition, (b) the occurrence of 1-methyladenosine in the T loop, a modification not present in the structurally related tRNA3cA,, and (c) the unusual presence of an unmodified uridine in the first position of the anticodon, which may be related to the unusual coding properties reported for this tRNA.

A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes

“…One of the subgroups is restricted to Yarrowia, Pachysolen, and the "Pichiaceae"; the other is common to all yeasts and contains the Saccharomyces tRNA Leu UAG . The latter is unique because it is the only Saccharomyces cerevisiae tRNA with an unmodified uridine in the wobble position of the anticodon triplet (Randerath et al 1979;Johansson and Byström 2005). Unmodified U34s have otherwise only been found in mitochondria, chloroplasts, and Mycoplasma species.…”

Ribosomal frameshifting in the yeast retrotransposon Ty: tRNAs induce slippage on a 7 nucleotide minimal site

“…Yeast is unusual in that it encodes a tRNA, tRNAf& (previously known as tRNA:B") that decodes all six leucine codons (Weissenbach et al, 1977). The expanded recognition requires an unmodified uracil at the wobble position of the anticodon (Randerath et al, 1979). Frameshifting could involve slippage of this tRNA between the two leucine codons.…”

An early step in wobble uridine tRNA modification requires the Elongator complex

“…Of these 13 tRNAs, the identity of the nucleoside at position 34 is known in eight species, and of these, six are mcm 5 U or ncm 5 U derivatives (Smith et al 1973;Kobayashi et al 1974;Kuntzel et al 1975;Yamamoto et al 1985;Keith et al 1990;Glasser et al 1992). The two others have an unmodified U (tRNA Leu UAG ) and a pseudouridine (⌿, tRNA Ile ⌿A⌿ ) at position 34 (Randerath et al 1979;Szweykowska-Kulinska et al 1994). The nature of the U 34 nucleoside in the five remaining tRNAs had not been established.…”