Figure 8.
The intact K108 residue of Rps3 is important for Cys-rti-tRNA incorporation at the UGA stop codon in the +4 base-specific manner. (A–G) The rps3Δ strains ME166 bearing the indicated rps3 mutant alleles on a single copy plasmid were introduced with the indicated dual luciferase readthrough reporter constructs (KP22 for UGA-C, KP35 for UGA-G, KP34 for UGA-A, KP36 for UGA-U, ZP40 for UAG-C and KP41 for UAA-C) in combination with either empty vector (EV) or indicated rti-tRNA on high copy plasmid, grown in SD and processed for stop codon readthrough measurements as described in Figure 2B. Schematics of individual rti-tRNAs base-pairing with the indicated stop codon tetranucleotides are given to the right of the plots; only the nucleotides of the anticodon loop are shown. (H) The readthrough stimulatory effect of the K108 residue requires proper conformation of the decoding center. The rps3Δ strains KPH126, 127 and 128 bearing the indicated rps3 mutant alleles on a single copy plasmid were grown in SD media without or with 400 μg/ml paromomycin for six hours and processed for stop codon readthrough measurements as described in Figure 2B. Statistical significance determined by the Student’s t-test is indicated (P< 0.05).