The effect of nucleotide composition at the end of the coding sequence on ribosomal read-through and translation re-initiation of a downstream gene.

Our computational analyses suggest that sequences which dictate features such as the local mRNA folding and the interaction of the rRNA and mRNA, when positioned in the end of an open reading frame (ORF), affect the fidelity of translation termination of that ORF and of downstream ones.

To understand and evaluate the strength of the effect of these features on translation termination we designed a plasmid that contains two fluorescent genes connected by a short linker sequence with no expression impact. The two genes, RFP followed by a GFP are transcribed from a single promoter. The 40 last nucleotides of the RFP were changed to have 3 ranges of Local Folding Energy (LFE), low, medium and high. The last 20 nucleotides within the previous 40 were changed to have no 16S rRNA binding, medium or high strength of binding to the anti- Shine-Dalgarno motif (Hybridization Energy (HE)). Overall, variants with 9 combinations were designed, all with synonymous nucleotide changes. These variants were generated and the resulted plasmids were transformed into E. coli to create 10 variant clones, including the original sequence.

The expression of both the RFP and GFP genes were examined. We show that while the RFP expression was affected mostly by the Local Folding Energy at the end of the RFP ORF, The downstream GFP expression was affected by both the LFE and the Hybridization Energy, where strong LFE and strong HE reduced the GFP expression significantly.