Use of mRNA Secondary Structure to Fine-Tune and Regulate Translation in Chloroplasts

Chloroplasts enable the production of very high amounts of heterologous proteins, up to 70% of total soluble protein. In addition, chloroplasts harbour many metabolic pathways of interest for synthetic biology approaches. It is therefore important to understand what influences protein levels and how gene expression is regulated. A major determinant is translation. The efficiency of translation initiation depends on start codon recognition. Start codons are defined by Shine-Dalgarno-sequences and/or local minima of mRNA secondary structure. Based on in vitro data it was hypothesised that translation initiation is regulated by RNA binding proteins that modify the mRNA secondary structure of the translation initiation region and thereby its accessibility. We tested this hypothesis by analysing in vivo mRNA secondary structure using two complementary methods (SHAPE-seq and DMS-MaPseq) and by analyzing translation by ribosome profiling. The Shine-Dalgarno sequence and the start codon of psbA, whose translation is upregulated in high light, are more accessible in conditions, which induce translation of psbA. Furthermore, we detected a footprint of a regulatory protein in the 5 UTR of psbA. It is such located that its binding can open the structure of translation initiation region and thereby making it accessible for the ribosome. Moreover, also other genes with weak Shine-Dalgarno sequences are regulated like psbA, while those with strong Shine-Dalgarno sequences are not. This indicates that the modulation of the accessibility of the translation initiation region by RNA binding proteins is a general mechanism to regulate translation initiation in plastids. These results highlight the importance of optimising the mRNA secondary structure of the gene of interest to obtain the desired protein levels.