Abstract
Shine-Dalgarno (SD) motifs are thought to play an important role in translational initiation in bacteria. Paradoxically, ribosome profiling studies in E. coli show no correlation between the strength of an mRNA's SD motif and how efficiently it is translated. Performing profiling on ribosomes with altered anti-Shine-Dalgarno sequences, we reveal a genome-wide correlation between SD strength and ribosome occupancy that was previously masked by other contributing factors. Using the antibiotic retapamulin to trap initiation complexes at start codons, we find that the mutant ribosomes select start sites correctly, arguing that start sites are hard-wired for initiation through the action of other mRNA features. We show that A-rich sequences upstream of start codons promote initiation. Taken together, our genome-wide study reveals that SD motifs are not necessary for ribosomes to determine where initiation occurs, though they do affect how efficiently initiation occurs.
Highlights
Translational initiation is a critical step in the regulation of gene expression that impacts which proteins are synthesized and to what extent
We show that A-rich sequences recently identified by Fredrick and co-workers (Baez et al, 2019) are enriched at annotated start sites compared to other AUG codons in the transcriptome where initiation does not take place; these A-rich sequences are found upstream of start codons in a wide variety of species across the bacterial kingdom
To perturb the function of SD motifs at the global level, we developed a new approach in which we mutate the ASD in 16S rRNA, purify the mutant ribosomes, and use ribosome profiling to ask how efficiently they translate each mRNA in the cell
Summary
Translational initiation is a critical step in the regulation of gene expression that impacts which proteins are synthesized and to what extent. Stable secondary structures surrounding the initiation site prevent 30S recruitment (Hall et al, 1982; de Smit and van Duin, 1990). The kinetics of RNA folding and unfolding are critical (de Smit and van Duin, 2003; Espah Borujeni and Salis, 2016): some structures exist in an unfolded state for such a short period of time that 30S subunits cannot find the start codon quickly enough by diffusion alone. In several well-characterized examples, regions of single-stranded RNA known as standby-sites are found nearby, positioning 30S subunits in close proximity so that they can efficiently capture the start codon upon unfolding of the mRNA secondary structure (de Smit and van Duin, 2003; Espah Borujeni et al, 2014).
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