EXPRESSION OF LACZ FROM THE PROMOTER OF THE ESCHERICHIA-COLI SPC OPERON CLONED INTO VECTORS CARRYING THE W205 TRP-LAC FUSION

The expression of lacZ has been analyzed and compared in a series of p romoter cloning vectors by measuring the amount of lacZ mRNA by hybrid ization and the amount of beta-galactosidase by standard enzymatic ass ay, Expression was driven by the promoter, P-spc, of the spc ribosomal protein operon, The vectors contained either the standard W205 trp-la c fusion with the trp operon transcription terminator, trpt, located i n the lacZ leader sequence, or a deletion derivative that functionally inactivates trpt, In the presence of trpt, lacZ expression was temper ature dependent so that increasing the growth temperature reduced the accumulation of lacZ mRNA and beta-galactosidase activity. The frequen cy of transcript termination at trpt was estimated to be near zero at 20 degrees C and at about 45% at 37 degrees C. The amount of P-spc-der ived lacZ mRNA and the amount of beta-galactosidase produced per lacZ mRNA varied, depending on the mRNA 5' leader sequence between P-spc an d lacZ. These results demonstrate that the quantitative assessment of promoter activities with promoter cloning vectors requires careful ana lysis and interpretation. One particular construct without trpt did no t seem to contain fortuitous transcription or translation signals gene rated at the fusion junction, In this strain, lacZ expression from P-s pc was compared at the enzyme activity and mRNA levels with a previous ly constructed strain in which lacZ was linked to the tandem P1 and P2 promoters of the rrnB operon, At any given growth rate, the different activities of beta-galactosidase in these two strains were found to r eflect the same differences in their amounts of lacZ mRNA, Assuming th at the promoter-lacZ fusions in these strains reflect the properties o f the promoters in their normal chromosomal setting, it was possible t o estimate the absolute transcription activity of P-spc and the relati ve translation efficiency of P-spc-lacZ mRNA at different growth rates . Transcription from the spc promoter was found to increase from about 10 transcripts per min at a growth rate of 1.0 doublings/h to a maxim um plateau of about 23 transcripts per min at growth rates above 1.5 d oublings/h. The translation frequency of lacZ mRNA expressed from P-sp c was unaffected by growth rates.