Regions of DNA involved in the stringent control of plasmid-encoded rRNA in vivo

Abstract

We have examined the transcription of two plasmid-encoded, stable RNAs; a shortened 16S ribosomal RNA and the spacer transfer RNA2Glu from the Escherichia coli rrnB operon. Plasmid deletions were constructed in vitro, in order to examine the DNA regions required for stringent control of rRNA expression in vivo during amino acid starvation. We find that rRNA synthesized from plasmids does exhibit a relA-dependent, stringent response. The DNA sequences required for this regulation do not extend beyond 20 bases downstream of the P1 transcription initiation site. Deletion of P2, the second of the two tandem rRNA promoters, does not weaken the stringent control of transcripts from P1. These results demonstrate that pause sites for RNA polymerase identified in vitro do not play a significant role in the stringent control of rRNA synthesis in vivo and imply that stringent regulation takes place at the level of initiation, rather than elongation, of transcription. Surprisingly, we find that the presence of extra intact rrnB operons (carried by a multicopy plasmid) reduces the magnitude of the stringent response.