We have used NMR to study the structure of the yeast tRNAphe sequence which was synthesized by using T7 RNA polymerase. Many resonances in the imino 1H spectrum of the transcript have been assigned, including those of several tertiary interactions. When the Mg2+ concentration is high, the transcript appears to fold normally, and the spectral features of the transcript resemble those of tRNAphe. The transcript has been shown to be aminoacylated with kinetics similar to the modified tRNAphe [Sampson, J. R., & Uhlenbeck, O. C.(1988) Proc, Natl. Acad. Sci. U.S.A. 85,1033-1037], suggesting that the structure of the two molecules must be similar. In the absence of Mg2+ or at [tRNA]:[Mg2+] ratios <0.2, the transcript does not adopt the native structure, as shown by both chemical shifts ana NOE patterns. In these low Mg2+ conditions, a second GU base pair is found, suggesting a structural rearrangement of the transcript. NMR data indicate that the structure of a mutant having G20 changed to U20 is nearly identical with that of the normal sequence, suggesting that the low aminoacylation activity of this variant is not due to a substantially different conformation.