Analysis of the regulatory sequences needed for induction of the chloramphenicol acetyltransferase gene cat-86 by chloramphenicol and amicetin.
Induction of the chloramphenicol acetyltransferase gene cat-86 in Bacillus subtilis results from the activation of translation of cat-86 mRNA. The inducers, chloramphenicol and amicetin, are thought to enable ribosomes to destabilize a stem-loop structure in cat-86 mRNA that sequesters the ribosome binding site for the cat-86 coding sequence, designated RBS-3. The region of cat-86 mRNA which is 5' to the stem-loop contained two additional ribosome binding sites, RBS-1 and RBS-2, located 84 and 56 nucleotides, respectively, upstream from RBS-3. RBS-1 and RBS-2 were each followed by a potential translation initiation codon and a short open reading frame. Bal 31-generated deletions into the 5' end of the regulatory region that removed RBS-1 but did not enter RBS-2 caused a fourfold decrease in the uninduced and chloramphenicol-induced level of cat-86 expression and a more than 10-fold reduction in the amicetin-induced level of expression. Deletions that removed both RBS-1 and RBS-2 but did not enter the stem-loop abolished both chloramphenicol- and amicetin-inducible expression. These data indicate that RBS-2 and sequences 3' to RBS-2 are minimally essential to chloramphenicol induction. However, the presence of RBS-1 in the mRNA elevated the maximum level of expression obtained during chloramphenicol induction. These studies also demonstrate that induction of cat-86 by amicetin is highly dependent on RBS-1. To determine whether a correlation existed between RBS-1 and amicetin inducibility, we examined the sequence of the regulatory regions for two natural variants of cat-86, cat-66 and cat-57, which are chloramphenicol inducible but are very poorly induced by amicetin. Both contained nucleotide sequence differences from cat-86 in the vicinity of RBS-1 that would prevent translation of the leader peptide associated with RBS-1 in cat-86. In contrast, the regulatory regions got the three genes were virtually identical in the vicinity of RBS-2. These data indicate that efficient induction by amicetin requires sequences that are not essential for induction by chloramphenicol.
Ambulos
N P
NP
Duvall
E J
EJ
Lovett
P S
PS
D000123:Acetyltransferases / Q000096:biosynthesis / Q000235:genetics*; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D002701:Chloramphenicol / Q000494:pharmacology*; D015500:Chloramphenicol O-Acetyltransferase; D004790:Enzyme Induction / Q000187:drug effects; D005809:Genes, Regulator / Q000187:drug effects*; D008969:Molecular Sequence Data; D011741:Pyrimidine Nucleosides / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; D011741:Pyrimidine Nucleosides; D011994:Recombinant Proteins; C004413:amicetin; D002701:Chloramphenicol; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase