Determination of the optimal aligned spacing between the Shine – Dalgarno sequence and the translation initiation codon of Escherichia coli m RNAs

TLDR

The Shine–Dalgarno sequence is essential for ribosome recognition of the translation start site, and its spacing relative to the AUG codon strongly influences translational efficiency. The study aimed to determine whether a unique optimal spacing exists between the SD sequence and the initiation codon. The authors inserted two series of synthetic RBSs with varying spacings and SD sequences into a plasmid carrying the chloramphenicol acetyltransferase gene, avoiding secondary structure. Protein expression measurements revealed a unique optimal aligned spacing of 5 nucleotides, corresponding to the distance between the 3′‑end of the 16S rRNA and the ribosomal P‑site.

Abstract

The prokaryotic mRNA ribosome binding site (RBS) usually contains part or all of a polypurine domain UAAGGAGGU known as the Shine – Dalgarno (SD) sequence found just 5′ to the translation initiation codon. It is now clear that the SD sequence is important for identification of the translation initiation site on the mRNA by the ribosome, and that as a result, the spacing between the SD and the initiation codon strongly affects translational efficiency (1). It is not as clear, however, whether there is a unique optimal spacing. Complications involving the definition of the spacing as well as secondary structures have obscured matters. We thus undertook a systematic study by inserting two series of synthetic RBSs of varying spacing and SD sequence into a plasmid vector containing the chloramphenicol acetyltransferase gene. Care was taken not to introduce any secondary structure. Measurements of protein expression demonstrated an optimal aligned spacing of 5 nt for both series. Since aligned spacing corresponds naturally to the spacing between the 3′-end of the 16s rRNA and the P-site, we conclude that there is a unique optimal aligned SD– AUG spacing in the absence of other complicating issues.