Chloramphenicol-induced translation of cat-86 mRNA requires two cis-acting regulatory regions.
Sequences essential to the chloramphenicol-inducible expression of cat-86, a chloramphenicol acetyltransferase gene, reside in a 144-base pair (bp) regulatory region that intervenes between the cat-86 coding sequence and its promoter. A key regulatory element within the 144-bp segment consists of a pair of inverted-repeat sequences that immediately precede the cat-86 coding region and span the ribosome-binding site for the gene. Because of the location of the inverted repeats, cat-86 transcripts are predicted to sequester the ribosome-binding site in a stable RNA stem-loop structure which should block translation of cat-86 mRNA. Chloramphenicol induction of gene expression is believed to result from ribosome-mediated destabilization of the RNA stem-loop structure, which frees the cat-86 ribosome-binding site, thereby allowing translation. In this study we demonstrated that deletion of 85 bp from the 5' end of the 144-bp regulatory region abolishes inducible expression of cat-86, although the gene is transcribed. This deletion leaves intact both the inverted repeats and the cat-86 coding sequence, and the deletion mutation is not complementable. Therefore, inducible regulation of cat-86 requires the inverted repeats plus an upstream, cis-acting regulatory region. The cis-acting region is believed to control translation of cat-86 mRNA by its essential participation in chloramphenicol-induced opening of the RNA stem-loop. cat-86 deleted for the 85-bp regulatory region and therefore virtually unexpressed was used to select for mutations that restore expression to the gene. An analysis of one mutant plasmid showed that the cat-86 gene is constitutively expressed and that this results from a duplication of the DNA sequence that spans the ribosome-binding site. The duplication provides cat-86 with two ribosome-binding sites. One of these sites is predicted to be sequestered in an RNA stem-loop, and the other is not involved in RNA secondary structure.
Ambulos
N P
NP
Mongkolsuk
S
S
Kaufman
J D
JD
Lovett
P S
PS
D000123:Acetyltransferases / Q000096:biosynthesis / Q000235:genetics*; D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D001665:Binding Sites; D002701:Chloramphenicol / Q000494:pharmacology*; D015500:Chloramphenicol O-Acetyltransferase; D004352:Drug Resistance, Microbial; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005809:Genes, Regulator; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic
D012329:RNA, Bacterial; D012333:RNA, Messenger; D002701:Chloramphenicol; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase