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.
