Mechanism of NusG-stimulated pausing, hairpin-dependent pause site selection and intrinsic termination at overlapping pause and termination sites in the Bacillus subtilis trp leader

Abstract

The Bacillus subtilis trpEDCFBA operon is regulated by TRAP-dependent transcription attenuation and translation repression mechanisms. Previous results showed that NusA and NusG cooperatively stimulate RNA polymerase pausing at U107 and U144 in the trp leader, and that NusG is required for pausing at U144 in vivo. Pausing at U107 and U144 participate in the attenuation and translation repression mechanisms, respectively, by providing additional time for TRAP binding. The intrinsic trp leader terminator overlaps the hairpin-dependent U144 pause site. Here, we conducted a systematic mutational analysis of the terminator/pause region. Deletion of the hairpin reduced pausing but did not affect pause site selection. Thus, hairpin-stimulated pausing is a more appropriate term than hairpin-dependent pausing for this pause site. In contrast, minor changes to the hairpin abolished termination. Sequences in the U-rich/T-rich tract following the hairpin affected termination and pausing differentially. The distance between the hairpin and the 3' end of the RNA dictates the position of termination, whereas the sequence downstream from the hairpin is responsible for pause site selection. NusA was found to increase both pausing and termination by reducing the rate of transcription. We also found that NusG-stimulated pausing is sequence specific and that NusG does not affect termination.