There are two major components of Escherichia coli ribosomes directly involved in selection and binding of mRNA during initiation of protein synthesis-the highly conserved 39 end of 16S rRNA (aSD) complementary to the ShineDalgarno (SD) domain of mRNA, and the ribosomal protein S1. A contribution of the SD-aSD and S1-mRNA interactions to translation yield in vivo has been evaluated in a genetic system developed to compare efficiencies of various ribosome-binding sites (RBS) in driving b-galactosidase synthesis from the single-copy (chromosomal) lacZ gene. The in vivo experiments have been supplemented by in vitro toeprinting and gel-mobility shift assays. A shortening of a potential SD-aSD duplex from 10 to 8 and to 6 bp increased the b-galactosidase yield (four-and sixfold, respectively) suggesting that an extended SD-aSD duplex adversely affects translation, most likely due to its redundant stability causing ribosome stalling at the initiation step. Translation yields were significantly increased upon insertion of the A/U-rich S1 binding targets upstream of the SD region, but the longest SD remained relatively less efficient. In contrast to complete 30S ribosomes, the S1-depleted 30S particles have been able to form an extended SD-aSD duplex, but not the true ternary initiation complex. Taken together, the in vivo and in vitro data allow us to conclude that S1 plays two roles in translation initiation: It forms an essential part of the mRNA-binding track even when mRNA bears a long SD sequence, and through the binding to the 59 untranslated region, it can ensure a substantial enhancing effect on translation.

Translation initiation in Escherichia coli involves as a rule complementary interactions between a Shine-Dalgarno (SD) sequence upstream of the initiation codon and a highly conserved 3'-end sequence of 16S rRNA (anti-SD). The translation efficiency is believed to be directly affected by the affinity of the ribosome to the mRNA initiation region. Earlier, high-affinity RNA ligands to E. coli ribosomes were selected by the SELEX approach, with the ligands containing an extended SD-sequence well represented. In this work, we examined the ability of artificial ribosome binding sites (RBSs) containing such an extended (10-nt) SD-sequence (superSD) to drive translation in vivo, as well as its ability to form the translation initiation complex in vitro. Toe print experiments showed the formation of a ternary initiation complex on mRNA comprising superSD. Moreover, they proved the formation of an extended SD-duplex in the binary 30S-mRNA complex. Nevertheless, the superSD appeared to be inefficient in translation in vivo. We believe that the initiation complex involving a superSD-element is too stable to be functional; it may impede the transition from initiation to elongation, thus disrupting the transcription-translation coupling and inhibiting the formation of polysomes.