Efficient Degradation of tRNA_i^Met May Involve Multiple Rounds of Deadenylation and Readenylation

Abstract

<p>Uncoupled exosome assay on pre-adenylated tRNA. 5′-end-labeled and in vitro polyadenylated unmodified tRNA_i^Met was incubated with 50 ng of affinity-purified Rrp6-TAP eluate alone or in combination with 50 ng of wild-type (Trf4-TAP) or mutant (DADA) complex or 50 ng of Trf4-TAP alone. Reactions were stopped after 30 (lanes 2, 6, 10, and 14), 60 (lanes 3, 7, 11, and 15), 90 (lanes 4, 8, 12, and 16), or 120 min (lanes 5, 9, 13, and 17) and separated on a 15% gel. The arrow indicates the position of the nonadenylated tRNA. Protein was omitted in lane 1. The migration positions of the polyadenylated tRNA (poly[A]) and the degradation products (dp) are indicated by brackets.</p