Translational initiation on structured messengers : another role for the Shine-Dalgarno interaction

Translational efficiency in Escherichia coli is in part determined by the Shine-Dalgarno (SD) interaction, i.e. the base-pairing of the 3′ end of 16 S ribosomal RNA to a stretch of complementary nucleotides in the messenger, located just upstream of the initiation codon. Although a large number of mutations in SD sequences have been produced and analysed, it has so far not been possible to find a clear-cut quantitative relationship between the extent of the complementarity to the rRNA and translational efficiency. This is presumably due to a lack of information about the secondary structures of the messengers used, before and after mutagenesis. Such information is crucial, because intrastrand base-pairing of a ribosome binding site can have a profound influence on its translational efficiency. By site-directed mutagenesis, we have varied the extent of the SD complementarity in the coat-protein gene of bacteriophage MS2. The ribosome binding site of this gene is known to adopt a simple hairpin structure. Substitutions in the SD region were combined with other mutations, which altered the stability of the structure in a predictable way. We find that mutations reducing the SD complementarity by one or two nucleotides diminish translational efficiency only if ribosome binding is impaired by the structure of the messenger. In the absence of an inhibitory structure, these mutations have no effect. In other words, a strong SD interaction can compensate for a structured initiation region. This can be understood by considering translational initiation on a structured ribosome binding site as a competition between intramolecular base-pairing of the messenger and binding to a 30 S ribosomal subunit. A good SD complementarity provides the ribosome with an increased affinity for its binding site, and thereby enhances its ability to compete against the secondary structure. This function of the SD interaction closely parallels the RNA-unfolding capacity of ribosomal protein S1. By comparing the expression data from mutant and wild-type SD sequences, we have estimated the relative contribution of the SD base-pairs to ribosome-mRNA affinity. Quantitatively, this contribution corresponds quite well with the theoretical base-pairing stabilities of the wild-type and mutant SD interactions.

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