FIGURE 1.

Systematic screen of the effects of synonymous codon repeats on firefly luciferase expression. (A) Schematic of reporter constructs used in these studies: Codon repeats (X) were inserted upstream of a firefly luciferase reporter gene either at position 4 of the coding region (X₄F), at position 314 in the Renilla-firefly fusion construct (RX₃₁₄F) or at position 4 of the coding region of Renilla luciferase (X₄R) or superfolder GFP (X₄G) (Pedelacq et al. 2006). X₄F and RX₃₁₄F were used for a systematic screen of the effects of 59 codons on expression. (B) Inhibitory effects of several codon repeats, but not CGA repeats, in X₄F are likely due to formation of strong RNA secondary structures. Luciferase activity for each 10-mer codon repeat at the N-terminus (%C) is plotted as a function of the free energy of the predicted RNA secondary structure from 1 to 50. Free energy of folding (nucleotides 1–50 of X₄F-reporter constructs containing 10 repeats of a particular codon for 59 of the 61 sense codons) (kcal/mol) was calculated using RNAstructure (Mathews et al. 1999) version 4.6 (http://rna.urmc.rochester.edu/RNAstructure.html). Similar results were obtained from calculations of free energy of folding from −4 to +37.