Development of a trpE promoter-strength measuring system and its use in comparison of the trpEDCBA, trpR and aroH promoters.
An expression system was developed for measuring in vivo promoter strength at the single copy level and this system was used to compare the trp, aroH and trpR promoters. This system employs trpE enzyme activity as a measure of promoter strength and lacZ expression for internal copy number reference. Promoter-containing fragments are inserted into a cloning vector and subsequently recombined on to phage lambda by genetic exchange. Single lysogens are then prepared and used in promoter-strength analyses. The strength of several promoters was determined using this system. Among the promoters tested, the Escherichia coli trpEDCBA promoter was the strongest; it was four times more active than the lacUV5 promoter and about ten times stronger than the trpR and aroH promoters. To validate measurement of trpE enzyme activity as an indicator of promoter strength, trpE enzyme activity was compared with the level of trpE mRNA. There was excellent correspondence between the two, suggesting that with this system trpE enzyme activity accurately reflects promoter strength. We also examined a homologous promoter-strength measuring system in which the promoter-cloning plasmid lacked a 104 base-pair DNA spacer that was present immediately downstream from the promoter-cloning site in our preferred system. We found that the spacer was essential; the transcribed region accompanying a cloned promoter apparently affected trpE translational efficiency and/or trpE mRNA stability.
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Department of Biological Sciences, Stanford University, CA 94305-5020.
Yanofsky
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D000878:Anthranilate Synthase / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D005798:Genes, Bacterial; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012706:Serratia marcescens / Q000235:genetics
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D000878:Anthranilate Synthase