Analysis of the secondary structure that negatively regulates inducible cat translation by use of chemical probing and mutagenesis.
Induction of chloramphenicol (Cm) acetyltransferase-encoding genes (cat) by Cm has been proposed to result from the destabilization of a stem-loop that sequesters the ribosome-binding site for the cat coding sequence. Destabilization is caused by the stalling of a ribosome at a specific site in the leader of cat transcripts that immediately precedes the stem-loop. By use of in vivo dimethylsulfate probing of cat-86 leader mRNA, we demonstrate the existence of the stem-loop structure in cat transcripts isolated from uninduced cells and its release during induction. Leader mutations chosen to provide the mRNA with an alternative folding pattern that destabilizes the stem-loop cause constitutive cat expression. Our results establish the occurrence in vivo of the stem-loop in cat-86 transcripts and its role as a negative regulator of cat expression.
Harrod
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R
Department of Biological Sciences, University of Maryland Baltimore County, Catonsville 21228.
Gu
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Lovett
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D001483:Base Sequence; D002701:Chloramphenicol / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D013728:Terminator Regions, Genetic
D012333:RNA, Messenger; D002701:Chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase