Regulation of the expression of the tufB operon: DNA sequences directly involved in the stringent control

We have located the DNA sequence involved in the stringent control of the Escherichia coli tufB operon. Various deletion and insertion mutants of the promoter locus were constructed by in vitro mutagenesis, and their response to guanosine-5'-diphosphate-3'-diphosphate (ppGpp) was examined in a cell-free transcription system consisting of purified RNA polymerase holoenzyme. The nucleotide sequence (GpCpGpC) from positions -7 to -4 (designating the initiation site of mRNA as position +1) is responsible for the selective inhibition by ppGpp of tufB transcription. Point mutations were then constructed in which each one of the above four nucleotides was replaced by an A or T residue and tested for their response to ppGpp in the in vitro transcription system. The results indicated that the alteration of any nucleotide in the GpCpGpC sequence leads to the loss of the stringent response.