FIGURE 6.

Measurement of the effect of −1 nt on Ψ-mediated nonsense suppression. (A) Western blot analysis. Total proteins were isolated from cells cotransformed with two constructs: (1) A TRM4 reporter construct, where a PTC (TAG, or UAG in RNA) was placed at codon 561 and the −1 nt (relative to the uridine of PTC) was A (lanes 1,5; original sequence), or changed to C (lane 2), G (lane 3) or T (or U in RNA) (lane 4), and (2) a PTC-specific designer guide RNA (lanes 2–5) or a nonspecific designer guide RNA (lane 1). After immunoprecipitation with antibody against protein A (part of the TAP tag at the carboxyl termini of Trm4 and Pro1), the proteins were resolved on SDS-PAGE, blotted, and probed with anti-Protein A antibody. The Trm4 PTC readthrough protein and the control protein (Pro1) are indicated. The RNA sequence surrounding the PTC (UAG at codon 561) is also shown (top). Capital letters indicate the PTC stop codon and N represents the −1 nt, which can be either A, C, G, or U. −1, +1, and +4 nt are indicated. Below the RNA sequence are the four possible codons when the −1 nt (N) is changed to A, C, G, or U, and the amino acids they code for. (B) Quantitation of the experiments shown in A. The intensity of Trm4 signal is normalized against Pro1 signal in the same lane, and compared with the signal (set as 1) of the original construct (the −1 nt is A) cotransformed with the PTC-specific guide RNA (Sample A561AΨAG, lane 5 in A).