Strong pauses at nucleotides +118 and +121 relative to the transcriptional start occur during in vitro transcription of the E. coli rnpB gene encoding the catalytic M1 RNA subunit of Ribonuclease P. These pauses are immediately downstream of 2 phylogenetically conserved stem-loop structures in the RNA. In the present work, single-base changes which disrupted Watson-Crick base-pairing in the hairpins were introduced into rnpB. Transcription studies in vitro with these modified templates revealed that none of the nucleotide changes predicted to increase or decrease the stability of the first hairpin significantly affected the pause half-lives. A mutation which disrupted the second hairpin increased the pause half-life 2-fold. The data suggest that the upstream stem and loop structures in the transcript are not involved in the pausing event.
