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. 2014 Jan;20(1):115–130. doi: 10.1261/rna.041467.113

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© 2013 Skowronek et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported), as described at http://creativecommons.org/licenses/by-nc/3.0/.

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FIGURE 2. — Yeast tRNA precursor is efficiently processed by recombinant Trz1. (A) Processing of an in vitro transcribed yeast nuclear pre-tRNA^Ser by 100 ng of recombinant Trz1. Lane p, processing reaction; lane c, control reaction without the protein. Precursor and products (mature tRNA and a 3′ trailer) are shown schematically on the right; dashed line denotes the intron in tRNA^Ser; DNA size marker (lane M, in nt) is shown on the left. (B) Determination of Trz1 cleavage site in vitro on pre-tRNA^Ser by primer extension. The major cleavage after the discriminator base and two additional, minor cleavages are indicated with arrows. A processing reaction was carried out with nonlabeled pre-tRNA, and the resulting 3′-trailer product was isolated and used as a template in the primer extension reaction. Sequencing reaction (lanes G,A,T,C) performed with the same primer (YC1) is shown on the left. The location of the primer relative to the tRNA is illustrated schematically at the bottom.