Analysis of the upstream region of the Escherichia coli rnd gene encoding RNase D. Evidence for translational regulation of a putative tRNA processing enzyme.
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 rho-independent transcription terminator. Surprisingly, deletion of the hairpin structure elevates rnd mRNA levels only slightly (less than 2-fold), but it dramatically decreases RNase D expression (greater than 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 expression is negatively regulated at the translational level by the initiation codon, and that an upstream structure (or sequence) also influences translation of this gene.
Zhang
J R
JR
Department of Biochemistry, University of Connecticut Health Center, Farmington 06032.
Deutscher
M P
MP
D001483:Base Sequence; D002872:Chromosome Deletion; D004722:Endoribonucleases / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D005809:Genes, Regulator; D008969:Molecular Sequence Data; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D043244:Ribonuclease III; D014158:Transcription, Genetic
D029968:Escherichia coli Proteins; D012333:RNA, Messenger; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli