Stringent regulation of transcription of the rnpB gene encoding the catalytic subunit of escherichia coli rNase P

Abstract

Escherichia coli RNase P, which catalyzed the processing of the 5``-termini of immature tRNAs, is composed of an RNA subunit (M1 RNA) and a protein subunit (C5 protein). The promoter region of the rnpB gene, which codes for M1 RNA, contains three promoter elements P-1, P-2, and P-3 in increasing distance from 5``-end of M1 RNA. In vivo and in vitro studies have shown that P-1 is the major site of transcription initiation of the rnpB gene. The P-1 promoter shares a consensus sequence with other promoters that can confer transcription repression upon the stringent response. The consensus sequence is located within the region (the discriminator region) between the -10 hexamer sequence and the transcription start site. The stringent response of the rnpB promoters was analyzed by directly observing metabolically unstable transcripts derived from the internally deleted rnpB gene harbored on a multicopy plasmid. Synthesis of the truncated transcripts was inhibited upon stringent condition induced by seryl-tRNA starvation, indicating that transcription of the rnpB gene is under negative stringent control. The inhibition of transcription was relA-dependent, which suggests that ppGpp is involved in the negative stringent response of rnpB transcription. Site-directed mutagenesis was carried out to examine the requirement of the rnpB promoter sequence for the stringent control. Mutant promoters were prepared by GC to AT substitutions at the discriminator region. The stringent response of the mutant promoters was examined both by analyzing synthesis of the truncated M1 RNA transcript upon seryl-tRNA starvation in vivo and by analyzing effects of ppGpp on in vitro transcription. The results showed that the discriminator region was responsible for the stringent control of the rnpB gene and the mutation of bases at different positions in the discriminator region differently affected the stringent response. Especially GC-rich sequences proximal to the transcription start site w...