Determination of the angle between the acceptor and anticodon stems of a truncated mitochondrial tRNA

Significant departures from the canonical (cloverleaf) secondary structure of transfer (t)RNAs can be found among the mitochondrial (m)tRNAs of higher metazoans; these mtRNAs thus pose a challenge to the concept of an invariant, L-shaped tertiary conformation for all tRNAs. For bovine mtRNA(Ser)(AGY), which lacks the entire 'dihydrouridine' (dhU) arm, two distinct tertiary models have been proposed: the first model preserves the L-shaped conformation at the expense of overall size; the second model preserves the absolute distance between the 3' terminus and the anticodon loop, while allowing the acceptor-anticodon interstem angle to vary. We have tested the central predictions of these two models by performing a series of transient electric birefringence measurements on bovine mtRNA(Ser)(AGY) constructs in which the aminoacyl-acceptor and anticodon stems were each extended by ~70 bp. This mtRNA species is particularly amenable to analysis, since the native bovine (heart) mtRNA is completely unmodified outside of the anticodon loop. For magnesium ion concentrations above 1 mM, the interstem angle for the extended mtRNA, 120 (±5)°, is approximately 50% larger than the corresponding angle for yeast tRNA(Phe) (70-80°) under the same ionic conditions. Furthermore, the interstem angles of the two tRNAs exhibit strikingly different responses to the addition of magnesium ions: the interstem angle for yeast tRNA(Phe) is reduced by nearly 50% upon addition of 2 mM magnesium ions, whereas the angle for mtRNA(Ser)(AGY) increases by about 10%. Our data thus support a central prediction of the second model; namely, that truncated mtRNAs will possess more open interstem angles. In addition, we demonstrate that birefringence amplitude data can be used to provide model-independent estimates for the interstem angles.