Portable Shine-Dalgarno Regions: A System for a Systematic Study of Defined Alterations of Nucleotide Sequences within E. coli Ribosome Binding Sites

We have developed a gene expression system in Escherichia coli that contains a portable Shine-Dalgarno region. Transcription of this system is under the direction of a hybrid promoter (tacII) derived from trp and lac-UV5 promoter sequences which is followed by a region that encodes a portable Shine-Dalgarno region (PSDR). Using a series of synthetic PSDRs, we varied the four bases that follow the Shine-Dalgarno (SD) region. We found that the presence of four A residues or four T residues in this position gives the highest translational efficiency. The presence of four C residues reduces the translation efficiency by 50% as compared with PSDRs with A or T residues. The presence of four G residues following the SD region lowers the translational efficiency by at least 75% with respect to PSDRs with A or T residues.