Control of translation efficiency in yeast by codon–anticodon interactions

The effects of CGA codons at amino acids 4 and 314 on full-length mRNA, examined by Northern analysis. (A) Diagram of the X₄R Renilla luciferase reporter indicating the position of the oligonucleotide probe used in panels B–D. Probe c is located at base pairs 682–705 in the 935 base-pair Renilla luciferase coding sequence. (B) Analysis of the effects of insertion of CGA codons at amino acid 4 upstream of Renilla luciferase on Renilla luciferase mRNA and activity. Renilla luciferase mRNA from X₄R reporter constructs containing RGS-(His)₆-(Arg)₈ was examined by Northern blots with the probe c indicated in panel A; U2 snRNA served as an internal control. Renilla luciferase mRNA levels were first normalized to levels of U2 snRNA and then compared to the wild-type Renilla luciferase mRNA containing RGS-(His)₆. In the lower panel, luciferase activity of Renilla luciferase reporter constructs containing RGS-(His)₆-(Arg)₈ insertions are shown. (Off, expression under repressing conditions.) Lanes 1–4 contain different amounts of input total RNA from the X₄R- RGS-(His)₆ (CGA)₈ bearing strain (20, 10, 5, 2.5 μg), while lanes 5–12 contain RNA (20 μg) from yeast strains bearing the indicated constructs. (C) Decoding of CGA codons with an anticodon-mutated tRNA^Arg species suppresses the CGA-mediated increase in Renilla luciferase mRNA. Northern blot analysis of Renilla luciferase mRNA from Renilla luciferase reporter constructs containing RGS-(His)₆-(Arg)₈, using Actin as an internal control. Strains expressed either a nonnatural exact match tRNA or a vector control. Luciferase mRNA amounts were normalized to the levels of Actin mRNA and then to RGS-(His)₆ (AGA)₈; In the titration indicated with the triangle, mRNA input from the strain X₄R- RGS-(His)₆ (CGA)₈ in a vector control was varied (20, 10, 5 μg). (D) Insertion of stop codons at amino acid 4 of Renilla luciferase resulted in a reduction in stable Renilla luciferase mRNA. Northern blot analysis of Renilla luciferase mRNA from indicated Renilla luciferase reporter constructs. Renilla luciferase mRNA was first normalized to levels of U1 snRNA and then to RLuc (wt) mRNA. (E) A diagram of the RX₃₁₄F reporter indicating the relative position of the probes used in panel F. Probe c is located at base pairs 682–705 in Renilla luciferase (935 base pairs), while probe d is located at base pairs 1564–1593 in the RX₃₁₄F reporter (2631 base pairs); this corresponds to base pairs 601–630 in firefly luciferase (1668 base pairs). Probe sequences are shown in Supplemental Table S3. (F) Analysis of the effects of insertion of CGA codons at amino acid 314 in the RX₃₁₄F reporter on Renilla-firefly luciferase mRNA. RNA from strains bearing RX₃₁₄F constructs with RGS-(His)_6, or RGS-(His)₆-(Arg)₈ encoded with either (AGA)₈, [(CGA)₄(AGA)₄], or (CGA)₈ as indicated was examined by Northern blots with the probes indicated in panel E. U2 snRNA served as an internal control.