Pausing and attenuation of in vitro transcription in the rrnB operon of E. coli

Abstract

We have determined the effects of the nusA gene protein and the regulatory nucleotide guanosine tetraphosphate (ppGpp) on pausing and termination of transcription in the leader region of the rrnB operon in vitro. The leader region of rrnB contains several types of potential regulatory sequences that act at the level of RNA chain elongation and may be involved in control of bacterial growth. We have mapped a termination site, tL, located 260 bases from rrnB promoter P1. Termination at tL is dependent on the nusA protein and is enhanced by ppGpp. The DNA sequence at tL shows striking homologies with trp t', a terminator also strongly affected in vitro by the nusA protein. These in vitro results suggest that rRNA transcription in vivo may be regulated in part through an attenuation mechanism that leads to termination of rrnB chains in the leader region. In addition to tL, elongation of transcription in the leader region is affected by several pause sites that are sensitive to the concentration of ppGpp and the presence of the nusA gene protein. The location and properties of these pause sites suggest that they may also play a role in regulation of rrnB transcription through a mechanism we have termed "turnstile" attenuation. One pause site, located 90 and 91 bases from P1, is unique in that it is not normally a site for transcriptional pausing, but is dependent on simultaneous binding of polymerase at rrnB promoter P2. This leads to blockage of P1 transcripts at high RNA polymerase densities and may provide an additional locus for regulation of rrn transcription.