Influence of the GCGC discriminator motif introduced into the ribosomal RNA P2- and tac promoter on growth-rate control and stringent sensitivity

Abstract

The synthesis of stable RNA in bacteria is known to be regulated by a stringent control mechanism. Characteristic of stringent-regulated promoters, all ribosomal RNA promoters P1, but not P2, contain a GC-rich discriminator sequence assumed to be important for such a control. Using site-directed mutagenesis we have altered both the rrnB P2 and the synthetic tac promoter to the consensus GCGC discriminator motif. The modified promoters were placed upstream of the structural gene encoding the chloramphenicol acetyltransferase. The response of the modified promoters to amino acid starvation, changes in the growth rate or differences in the basal level of guanosine tetraphosphate (ppGpp) were determined in vivo. The results clearly show, that the discriminator motif is sufficient to convert the ribosomal RNA promoter P2 to a stringent, as well as growth-rate regulated, promoter. By contrast, the same discriminator sequence linked to the synthetic tac promoter does not convert this promoter to either stringency or growth-rate regulation. Finally, the results presented in this study reinforce the view that stringent and growth-rate regulation utilize the same mechanism, with ppGpp being the common mediator.