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

摘要: We have investigated the specificity of eukaryotic enzymatic machinery that transforms adenosine at position 37 (39 adjacent to anticodon) several tRNAs into threonylcarbamoyladenosine (t 6 A37). To this end, 28 variants yeast initiator tRNA Met and Val , devoid modified nucleotide, were produced by in vitro transcription with T7 polymerase corresponding synthetic genes microinjected cytoplasm Xenopus laevis oocytes. Threonylcarbamoyl incorporation was analyzed transcripts mutated anticodon loop substitution, deletion, or insertion nucleotides, overall 3D structure altering critical tertiary interactions. Specifically, we tested effects ribonucleotides loop, changes size, perturbations due mutations disruptive base pairs, truncated tRNAs. The results indicate that, addition targeted A37, only U36 absolutely required. However, A38 considerably facilitates quantitative conversion A37 t catalyzed enzymes present X. laevis. positions 34 35 “neutral” can accept any four canonical nucleotides A, U, C, G. size may vary from six eight stem one mismatch pair type Ap Co r G pU location 30‐40 without affecting efficiency formation still is efficiently formed. Although threonylcarbamoylation occurred having limited structure, L-shaped architecture substrate required for efficient A37. These favor idea unique located oocyte catalyzes all U36A37-containing (anticodon NNU). Microinjection Meti oocytes also revealed activities other nucleotide modifications, respectively m 1 G9 ,m 2 10 26 7 46 ,D 47 5 C 48/49, A58.