Nucleotide-specific transcriptional pausing in the pyrBI leader region of Escherichia coli K-12

Abstract

Expression of the pyrBI operon, which encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase, is regulated primarily through a UTP-sensitive transcriptional attenuation control mechanism in Escherichia coli. In this mechanism, the extent of coupling between transcription and translation within the pyrBI leader region determines the level of p-independent transcriptional termination at an attenuator preceding the pyrB gene. A key feature in this mechanism is transcriptional pausing that occurs before the attenuator in the pyrBI leader region when UTP concentrations are low. In this study, we characterized in detail this UTP-sensitive transcriptional pausing in vitro. Our results show that pausing occurs, to different extents, before the addition of every uridine residue in the leader transcript. This pausing occurred only at low UTP concentrations (20-200 microM) that cause high levels of pyrBI expression in vivo. Transcriptional pausing did not occur in the pyrBI leader region at 20 microM CTP or ATP and only occurred strongly at one site at 20 microM GTP. We also demonstrated that NusA functions as a general enhancer of UTP-sensitive transcriptional pausing in the pyrBI leader region. Finally, we identified at the 3' end of the pyrBI attenuator a 3-base region at which termination occurs. Selection of termination sites within this region was shifted by varying the UTP concentration or including NusA in the transcription reaction mixture.