The Trf4 Complex Preferentially Polyadenylates Unmodified tRNA_i^Met and the Unmodified A34GΔU13 Mutant of tRNA^Ala

Abstract

<div><p>(A) Polyadenylation assay with Trf4p-TAP and unmodified and native tRNA_i^Met as substrates. The 5′-end-labeled tRNAs were incubated with 50 ng of Trf4 complex for times indicated and resolved by gel electrophoresis. The migration position of the input tRNA is indicated by an arrow.</p> <p>(B) Sequence of yeast tRNA_i^Met in cloverleaf form with all the known nucleotide modifications. Tertiary interactions are indicated in solid and dashed red lines. The network of hydrogen bonds involving A20, G57, m¹A58, A59, and A60, necessary and unique for tertiary interactions in eukaryotic initiator tRNA, is outlined by solid red lines. A*, 5′-phosphoribosyl-2′-adenosine; D, dihydrouridine; m¹A, 1-methyladenosine; m¹G, 1-methylguanosine; m²G, <i>N²</i>-methylguanosine; m²₂G, <i>N²,N²</i>-dimethylguanosine; m⁷G, 7-methylguanosine; t⁶A, <i>N⁶</i>-threonylcarbamoyladenosine.</p> <p>(C) Polyadenylation activity of Trf4p-TAP on unmodified wild-type, A34GΔU13 mutant tRNA^Ala, and native tRNA^Ala. The 5′-end-labeled tRNAs were incubated with 20 or 50 ng of Trf4 complex for 30 min at 30 °C. Lane I in each panel shows input tRNA. tRNAs were separated on 10% denaturing gels.</p> <p>(D) Yeast tRNA^Ala in its cloverleaf form. All known nucleotide modifications are indicated. In the mutant tRNA^Ala used in this study, the uracil at position 13 was deleted, and the A34 in the anticodon loop was changed to guanine (marked in red). ψ, pseudouridine; m¹I³⁷, 1-methylinosine generated by deamination of adenosine at position 37. Other modifications are labeled as in (B).</p></div