Figure 5.

Evidence that tRNA binding by Gcd14p/Gcd10p requires Gcd10p. The indicated purified FlagGcd14p/Gcd10p complexes (A and B) or FlagGcd14p alone (C) or comparable amounts of protein purified from untagged (GCD14, D) were combined in TB binding buffer with [³²P]tRNA_i^Met purified from the gcd10Δ hcIMT4 strain. Reactions were filtered on a slot blot apparatus containing a three-membrane sandwich, as described in Materials and Methods. The cpm of ³²P retained on the second membrane (nitrocellulose), which typically contains specific RNA-protein complexes, was quantified by phosphorimaging analysis using a Storm 860 apparatus and imagequant software. The graph shows the relative amount of bound [³²P]tRNA_i^Met plotted against the protein concentration for each reaction, with columns 1–5 designating reactions containing protein concentrations of 0, 8, 16, 32, or 64 nM, respectively. (Inset) An autoradiogram of the nitrocellulose membrane used to generate the graphical data. The rows A–D and columns 1–5 designate the identities of the complex and protein concentrations, respectively, as described in the graph.