Two hybrid promoters that are functional in Escherichia coli have been constructed. These hybrid promoters,, tad and tacd, were derived from sequences of the trp and the lac UV5 promoters. In the. first hybrid promoter (tacT), the DNA upstream ofposition -20 with respect to the transcriptional start site was derived from the trp promoter. The DNA downstream of position -o20 was derived. from the iac UV5-promoter. In the second hybrid promoter (tacH), the DNA upstream ofposition -11 at the Hpa 1 site within the Pribnow box was derived from the trp promoter. The DNA downstream of position -11 is a 46-base-pair synthetic DNA fragment that specifies part ofthe.hybrid Pribnow box and the entire lac operator. It also specifies a Shine-Dalgarno sequence flanked by two unique restriction sites (portable ShineDalgarno sequence). The-tacd and the tacll promoters respectively direct transcription approximately 11 and 7 times more efficiently than the derepressed parental lac UV5 promoter and approximately 3 and 2 times more efficiently than the trp promoter in the absence of the trp repressor. Both hybrid promoters can be repressed by the lac repressor and both can be derepressed with isopropyl -D-thiogalactoside. Consequently, these hybrid promoters are useful for the controlled expression of foreign genes at high levels in E. coli. In contrast to the trp and thelac UV5 promoters, the tad promoter has not only a consensus -35 sequence but also a consensus Pribnow box sequence. This may explain the higher efficiency of this hybrid promoter with respect to either one of the parental promoters.The DNA sequences of many prokaryotic promoters (reviewed in refs. 1-3) are known, yet very little is known about which features in these sequences determine the efficiency of promoters. Two domains upstream of the start site of transcription have been identified for which a consensus sequence has been formulated (1-5). These domains are the -35 sequence (5'-T-T-G-A-C-A) and the Pribnow box (5'-T-A-T-A-A-T) in the' -10 region. Both domains are in close contact with the RNA polymerase during initiation ofRNA synthesis (2,. 6). Almost all promoter mutations map in or near these domains (reviewed in ref. 1).The relative efficiencies of only a few promoters have been measured (7). We have determined the relative efficiencies of four promoters and we show that the efficiency of the lac UV5 promoter can be greatly increased by replacing its -35 region with the -35 region of the stronger trp promoter. Here, we describe the construction and the properties of the hybrid promoters tacT and tacIW.MATERIALS AND METHODS Bacterial Strains. For standard transformations Escherwhia coli 294 (ref. 8) was used routinely. The lac-repressor-overproducing strain used in this study was E. coli D1210, whichwas kindly provided by J. R. Sadler (9). This strain carries the ladq and lacY+ alleles on the chromosome but otherwise is identical to E. coli HB101 [F-lacId, lacO+, lacZ+, lacY-, gal-, pro, leu-, th, end-, hsm-, hsr-, recA-, rpsL-] from which it w...

In a lacZ expression vector (pMC1403Plac), all 64 codons were introduced immediately 3′ from the AUG initiation codon. The expression of the second codon variants was measured by immunoprecipitation of the plasmid‐coded fusion proteins. A 15‐fold difference in expression was found among the codon variants. No distinct correlation could be made with the level of tRNA corresponding to the codons and large differences were observed between synonymous codons that use the same tRNA. Therefore the effect of the second codon is likely to be due to the influence of its composing nucleotides, presumably on the structure of the ribosomal binding site. An analysis of the known sequences of a large number of Escherichia coli genes shows that the use of codons in the second position deviates strongly from the overall codon usage in E. coli. It is proposed that codon selection at the second position is not based on requirements of the gene product (a protein) but is determined by factors governing gene regulation at the initiation step of translation.

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.

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