The upstream region of the Escherichia coli rnd gene has been analyzed. A single promoter and transcription start site were identified in the 170-nucleotide upstream region of the clone based on deletion analysis, nuclease S1 mapping, primer extension, and quantitative dot-blots. The start site of transcription is 70 nucleotides upstream of the UUG initiation codon of the RNase D coding region. Between the RNA initiation site and the coding region is a GC-rich hairpin structure followed by 8 T residues that looks like a ρ-independent transcription terminator. Surprisingly, deletion of the hairpin structure elevates rnd mRNA levels only slightly (<2-fold), but it dramatically decreases RNase D expression (>95%), measured both by activity and immunoblotting. No evidence for an effect on mRNA stability or processing was observed. The uncommon UUG translation initiation codon was converted to AUG by site-directed mutagenesis. This alteration led to an 11-fold elevation of RNase D expression in single-copy plasmids. These data indicate that RNase D epxression is negatively regulated at the translational level by the initiation codon, and that an upstream structure (or sequence) also influences translation of this gene.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - Jan 1 1989|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology