It is widely accepted that mRNA sequences present near the start codon of a gene can influence gene expression, whether by direct ribosome binding, interaction with additional cellular factors, formation of secondary structure, or a combination of these activities. Although various methods have been used to study the effect of different sequences on expression, the lack of studies utilizing both exhaustive sampling and high-throughput sequencing has left gaps in our understanding. We have exploited the in vitro technique ribosome display and a minimal, reconstituted E. coli-based translation system to select fast-translating sequences from an mRNA library containing all possible 18-base sequences situated directly before the AUG start codon of a well-expressed, robustly-folding model protein. Several selection rounds of increasing stringency (i.e., shorter translation times) were performed to enrich the most efficient sequences. High-throughput sequencing on the Roche 454 platform was used to generate enough information to allow meaningful statistical analysis of the selected pools. As expected, a large number of Shine-Dalgarno sequences in various contexts were selected; however, the majority of sequences from the initial selection were highly cytosine-rich with no Shine-Dalgarno motifs. C-rich sequences are not abundant in E. coli but have been implicated as translation initiation sites in distantly related organisms. A k-motif search (k = 4-8) revealed that highly-represented motifs are often complementary to solvent-exposed portions of the 16S rRNA of the 30S ribosomal subunit. Single-clone ribosome display confirmed that several C-rich clones were highly efficient in the minimal in vitro system used for selection. This activity was sensitive, however, to the presence of competitor oligonucleotides, which supports our hypothesis of mRNA-rRNA base-pairing and explains why these sequences have no activity in E. coli. Ultimately, our results indicate that the nature of the ribosome, a broad-specificity ribozyme, allows for many solutions to the fast-translation problem, including some not currently utilized in vivo. Also, transient base-pairing between the 16S rRNA and the mRNA near the start codon seems to expedite initiation
