RNA determinants required for L4-mediated attenuation control of the S10 r-protein operon of Escherichia coli

Abstract

We have probed regions of the S10 leader RNA to determine their role in L4-mediated, NusA-dependent attenuation control of the S10 ribosomal protein operon. Using genetic and "antisense" oligonucleotide competition approaches, we were able to distinguish between the determinants necessary for intrinsic (NusA-independent) pausing by RNA polymerase at the S10 attenuation site, for NusA-dependent enhancement of pausing, and for L4 stabilization of the paused ternary complex. The upper stem-loop structure in the attenuator hairpin is the major determinant for the NusA-dependent pause, while the sequence at the site of pausing is important for RNA polymerase to pause in the absence of NusA. The determinants for L4 stabilization of the paused complex include the hairpin immediately upstream of the attenuator hairpin as well as the ascending side of the attenuator structure. In conclusion, our results suggest that there are three distinct pausing activities by RNA polymerase during its transcription of the S10 leader, with three corresponding signals in the S10 leader.