Activities of constitutive promoters in Escherichia coli

Abstract

The in vivo activities of seven constitutive promoters in Escherichia coli have been determined as functions of growth rate in wild-type relA+spoT+strains with normal levels of guanosine tetraphosphate (ppGpp) and in ppGpp-deficient ΔrelAΔspoT derivatives. The promoters include (i) the spc ribosomal protein operon promotor Pspc; (ii) the β-lactamase gene promotor Pblaof plasmid pBR322; (iii) the PLpromoter of phage λ; (iv) and (v) the replication control promoters PRNAIand PRNAIIof plasmid pBR322; and (vi) and (vii) the P1 and P2 promoters of the rrnB ribosomal RNA operon. Each strain carried an operon fusion consisting of one of the respective promoter regions linked to lacZ and recombined into the chromosome at the mal locus of alac deletion strain. The amount of 5′-terminal lacZ mRNA and of β-galactosidase activity expressed from these promoters were determined by standard hybridization or enzyme activity assays, respectively. In addition, DNA, RNA and protein measurements were used to obtain information about gene dosage, rRNA synthesis and translation rates. By combininglacZ mRNA hybridization data with gene dosage and rRNA synthesis data, the absolute activity of the different promoters, in transcripts/minute per promoter, was determined. In ppGpp-proficient (relA+spoT+) strains, the respective activities of rrnB P1 and P2 increased 40 and fivefold with increasing growth rate between 0.7 and 3.0 doublings/hour. The activities of Pspc, PL, Pbla, and PRNAIincreased two- to threefold and reached a maximum at growth rates above 2.0 doublings/hour. In contrast, PRNAIIactivity decreased threefold over this range of growth rates. In ppGpp-deficient (ΔrelAΔspoT) bacterial strains, the activities of rrnB P1 and P2 promoters both increased about twofold between 1.6 and 3.0 doublings/hour, whereas the activities of Pspc, PL, Pbla, and PRNAI, and PRNAIIwere about constant. To explain these observations, we suggest that the cellular concentration of free RNA polymerase increases with increasing growth rate; for saturation the P1 and P2 rRNA promoters require a high RNA polymerase concentration that is approached only at the highest growth rates, whereas the other promoters are saturated at lower polymerase concentrations achieved at intermediate growth rates. In addition, the data indicate that the respective rrnB P1 and PRNAIIpromoters were under negative and positive control by ppGpp. This caused a reduced activity ofrrnB P1 and an increased activity of PRNAIIduring slow growth in wild-type (relA+spoT+) relative to ppGpp-deficient (ΔrelAΔspoT) bacterial strains.