Site in the cat-86 regulatory leader that permits amicetin to induce expression of the gene.
Expression of the plasmid gene cat-86 is induced in Bacillus subtilis by two antibiotics, chloramphenicol and the nucleoside antibiotic amicetin. We proposed that induction by either drug causes the destabilization of a stem-loop structure in cat-86 mRNA that sequesters the ribosome-binding site for the cat coding sequence. The destabilization event frees the ribosome-binding site, permitting the initiation of translation of cat-86 mRNA. cat-86 induction is due to the stalling of a ribosome in a leader region of cat-86 mRNA, which is located 5' to the RNA stem-loop structure. A stalled ribosome that is active in cat-86 induction has its aminoacyl site occupied by leader codon 6. To test the hypothesis that a leader site 5' to codon 6 permits a ribosome to stall in the presence of an inducing antibiotic, we inserted an extra codon between leader codons 5 and 6. This insertion blocked induction, which was then restored by the deletion of leader codon 6. Thus, induction seems to require the maintenance of a precise spatial relationship between an upstream leader site(s) and leader codon 6. Mutations in the ribosome-binding site for the cat-86 leader, RBS-2, which decreased its strength of binding to 16S rRNA, prevented induction. In contrast, mutations that significantly altered the sequence of RBS-2 but increased its strength of binding to 16S rRNA did not block induction by either chloramphenicol or amicetin. We therefore suspected that the proposed leader site that permitted drug-mediated stalling was located between RBS-2 and leader codon 6. This region of the cat-86 leader contains an eight-nucleotide sequence (conserved region I) that is largely conserved among all known cat leaders. The codon immediately 5' to conserved region I differs, however, between amicetin-inducible and amicetin-noninducible cat genes. In amicetin-inducible cat genes such as cat-86, the codon 5' to conserved region I is a valine codon, GTG. The same codon in amicetin-noninducible cat genes is a lysine codon, either AAA or AAG. When the GTG codon immediately 5' to conserved region I in cat-86 was changed to AAA, amicetin was no longer active in cat-86 induction, but chloramphenicol induction was unaffected by the mutation. The potential role of the GTG codon in amicetin induction is discussed.
D000123:Acetyltransferases / Q000235:genetics*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D002701:Chloramphenicol / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase; D003062:Codon / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics; D011741:Pyrimidine Nucleosides / Q000494:pharmacology; D012045:Regulatory Sequences, Nucleic Acid; D012270:Ribosomes
D000900:Anti-Bacterial Agents; D003062:Codon; D021382:Protein Sorting Signals; D011741:Pyrimidine Nucleosides; C004413:amicetin; D002701:Chloramphenicol; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase