Abstract
We recently reported that a ribosome binding site (RBS) derived from gene 10 of bacteriophage T7 (g10-L) causes a pronounced stimulation of expression when placed upstream of a variety of genes, and that this effect is probably due to a stimulation of translation efficiency in Escherichia coli (Olins, P. O., Devine, C. S., Rangwala, S. H., Kavka, K. S. (1988) Gene (Amst.) 73, 227-235). Here we present a model for the mechanism of action of the g10-L: the RBS contains a 9-base sequence which has the potential for forming a novel base-paired interaction with bases 458-466 of the 16 S rRNA of E. coli. Although such sequence homologies are rare in E. coli RBS regions, a number of similar sequences were found in the RBS regions of other bacteriophage structural genes. When an isolated homology sequence was placed upstream of a synthetic RBS, there was a 110-fold increase in the translation efficiency of the lacZ gene. Surprisingly, the homology sequence also stimulated translation when placed downstream of the initiator codon, indicating that this sequence is acting as a translational "enhancer."
Highlights
Useful for expression of foreign genes in E. coli [17]
Missouri 63198 leader is largely due to the presence of a nine-base sequence upstream of the Shine-Dalgarno region
The resulting plasmid, pMON 5527, is illustrated in base sequence which has the potential for forming a novel base-paired interaction with bases 458-466 of fragments and substituted into pMON 5527 using the unique BglII, the 16 S rRNAof E. coli
Summary
Placed upstream of a variety of genes, and that this Plasmid Construction-DNA manipulations were performed aceffect is probably due to a stimulation of translation cording to Maniatis et al [17]. The resulting plasmid, pMON 5527, is illustrated in base sequence which has the potential for forming a. Quadruplicate samples of 10 pgof RNA were applied to filters and probed with 5’-32P-labeledDNA oligonucleotidesspecific for either the lac gene 5’-ATTTGTGTAGTCGGTTTA-3o’r)for E. coli 16S rRNA (having homologies, apart from the well established Shine-Dalgarno the sequence 5’-CCAGTAGTTATCCCCCTCCATCAGG-3’R)a.diosequence [4]. The search for sequence specificity in the initiation process has bleen unproductive; apart from the need activity was determined by measuring the Cernekov radiation present on the filters. For an initiator codon and Shine-Dalgarno sequence, it has been widely held that themajor determinants of the initiation
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