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. 2012 Feb 8;40(12):5679–5693. doi: 10.1093/nar/gks223

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© The Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Mutational analysis of Trf4p reveals minimal functional Trf4p and indicates the importance of D425 in PAP activity. (A) Fragment of Trf4p (aminoacids 161–481) used for previous structural studies does not support growth in trf4Δtrf5Δ background. Growth test analysis of wt and mutants of TRF4 episomally expressed in trf4Δtrf5Δ strain complemented with WT TRF4 on URA3 plasmid and serially diluted and spotted on media with 5-FOA. (B) Overview of mutant forms of TRF4 used in this study: CAT, catalytical domain; CD, central domain. (C) Minimal viable allele of TRF4 spans the amino acids 96–499. Drop tests were performed analogously to (A). (D) Northern blot analysis of total RNA isolated from WT and mutant TRF4 yeast strains with DNA probes specific for indicated RNAs. (E) In vitro polyadenylation assays with 20 ng affinity purified WT and mutant TRAMP4 were performed in the presence of 0.5 mM ATP, 10 ng radioactively labeled hypomodified tRNA_i^Met (migration indicated by an arrow). Reactions were stopped after 15, 30 and 60 min, respectively. The first lane (np) represents control sample with no protein added. RNAs were separated on 20% PAGE. (F) Coupled polyadenylation and exosome assay. The 5′-end-labeled unmodified tRNA_i^Met incubated with 40 ng of affinity-purified TRAMP4 (ProtA-Trf4p) and 40 ng of nuclear exosome (Rrp6-TAP) for 15, 30 and 60 min. The first lane (np) represents control sample with no protein added. (G) D425A mutation lowers Trf4p affinity to ATP. Polyadenylation assay was performed with 25 ng of affinity purified D425A Trf4p, 50 fmol 5′-end-labeled unmodified tRNA_i^Met in the presence of ATP concentrations indicated. Reactions were stopped after 60 min and resolved on 10% denaturing gel.