Effects of rare codon clusters on the expression of a high-turnover chloroplast protein in Chlamydomonas reinhardtii

Expression of foreign proteins in chloroplasts has become an important field of plant genetic engineering. Optimized codon usage is generally thought to increase translational efficiency, but high speed translation of codon bias-adjusted mRNAs can also result in protein misfolding due to a lack of rare codons. In order to analyze the effect of rare codons on a native chloroplast protein in vivo, we modified the D1 subunit of photosystem II by fusing small peptides with different codons into a loop region which tolerates insertions without loss of function. Because of its high-turnover properties, the D1 protein represents an excellent test object to investigate the impact of rare codons on its translation. We choose codons for amino acids Arg, Leu, Ser, Ala and Gly which are rarely used and compared translation of the modified D1 proteins with the respective mutant proteins containing insertions with frequently used codons. Our data indicate that only rare Arg codons drastically affect synthesis of the D1 protein and cluster of rare Ser-codon can induce strategic ribosomal pausing sites.