Influences on gene expression in vivo by a Shine–Dalgarno sequence

Summary

The Shine–Dalgarno (SD⁺: 5′-AAGGAGG-3′) sequence anchors the mRNA by base pairing to the 16S rRNA in the small ribosomal subunit during translation initiation. We have here compared how an SD⁺ sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD⁺ is confirmed. A downstream SD⁺ gives decreased gene expression. This effect is also valid for appropriately modified natural Escherichia coli genes. If an SD⁺ is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD⁺ is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD⁺ and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD⁺ located between them. A minor positive contribution to upstream initiation resulting from 3′ to 5′ ribosomal diffusion along the mRNA is suggested. Analysis of the E. coli K12 genome suggests that the SD⁺ or SD-like sequences are systematically avoided in the early coding region suggesting an evolutionary significance.