Structural requirements for enzymatic formation of threonylcarbamoyladenosine (t⁶A) in tRNA: An in vivo study with Xenopus laevis oocytes

Abstract

We have investigated the specificity of the eukaryotic enzymatic machinery that transforms adenosine at position 37 (3′ adjacent to anticodon) of several tRNAs into threonylcarbamoyladenosine (t⁶A₃₇). To this end, 28 variants of yeast initiator tRNA^Met and yeast tRNA^Val, devoid of modified nucleotide, were produced by in vitro transcription with T₇ polymerase of the corresponding synthetic tRNA genes and microinjected into the cytoplasm of Xenopus laevis oocytes. Threonylcarbamoyl incorporation was analyzed in tRNA transcripts mutated in the anticodon loop by substitution, deletion, or insertion of nucleotides, or in the overall 3D structure of the tRNA by altering critical tertiary interactions. Specifically, we tested the effects of altering ribonucleotides in the anticodon loop, changes of the loop size, perturbations of the overall tRNA 3D structure due to mutations disruptive of the tertiary base pairs, and truncated tRNAs. The results indicate that, in addition to the targeted A₃₇, only U₃₆ was absolutely required. However, A₃₈ in the anticodon loop considerably facilitates the quantitative conversion of A₃₇ into t⁶A₃₇ catalyzed by the enzymes present in X. laevis. The anticodon positions 34 and 35 were absolutely “neutral” and can accept any of the four canonical nucleotides A, U, C, or G. The anticodon loop size may vary from six to eight nucleotides, and the anticodon stem may have one mismatch pair of the type A∗C or G∗U at location 30–40 without affecting the efficiency of t⁶A₃₇ formation and still t⁶A₃₇ is efficiently formed. Although threonylcarbamoylation of A₃₇ occurred with tRNA having limited perturbations of 3D structure, the overall L-shaped architecture of the tRNA substrate was required for efficient enzymatic conversion of A₃₇ to t⁶A₃₇. These results favor the idea that unique enzymatic machinery located in the oocyte cytoplasm catalyzes the formation of t⁶A₃₇ in all U₃₆A₃₇-containing tRNAs (anticodon NNU).

Microinjection of the yeast tRNA^Meti into the cytoplasm of X. laevis oocytes also revealed the enzymatic activities for several other nucleotide modifications, respectively m¹G₉, m²G₁₀, m₂²G₂₆, m⁷G₄₆, D₄₇, m⁵C_48/49, and m¹A₅₈.