Summary: We have previously cloned and sequenced the gene, encoding inorganic pyrophosphatase (PPase), of K12 [Lahti, R., Pitkäranta, T., Valve, E., Ilta, I., Kukko-Kalske, E. & Heinonen, J. (1988) 170, 5901–5907]. In this work mutations were constructed in the 5′ flanking region of and the effect on expression was determined. The minimum length of the fully active 5′ flanking region was shown to be 117 bp. Further deletion decreased the activity, and upon deletion to nucleotide —37 the promoter activity was totally lost. A clear point of inflection was observed in the inactivation upon deletion over the nucleotide —50. This is consistent with the fact that by binding to promoters RNA polymerase holoenzyme generally covers the —50 to +20 region in genes. When the —35 sequence of , AAGACA, was mutated to AAAACA, expression, as measured by PPase production, decreased to 20% of the wild-type, whereas by the change of the —10 sequence, TATAAT, to TTTAAT or TATAAA, the gene was totally inactivated. Furthermore, when the ribosome-binding site (RBS) sequence, AGGAAA, was altered to AAGAAA, PPase production decreased to 19% of the wild-type. Surprisingly, when the RBS sequence was mutated to the consensus RBS sequence, AGGAGG, the intracellular levels of both mRNA and PPase decreased drastically. The implications of these results are discussed.


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