title: Complementarity of Bacillus subtilis 16S rRNA with sites of antibiotic-dependent ribosome stalling in cat and erm leaders.
abstract: Inducible cat and erm genes are regulated by translational attenuation. In this regulatory model, gene activation results from chloramphenicol- or erythromycin-dependent stalling of a ribosome at a precise site in the leader region of cat or erm transcripts. The stalled ribosome is believed to destabilize a downstream region of RNA secondary structure that sequesters the ribosome-binding site for the cat or erm coding sequence. Here we show that the ribosome stall sites in cat and erm leader mRNAs, designated crb and erb, respectively, are largely complementary to an internal sequence in 16S rRNA of Bacillus subtilis. A tetracycline resistance gene that is likely regulated by translational attenuation also contains a sequence in its leader mRNA, trb, which is complementary to a sequence in 16S rRNA that overlaps with the crb and erb complements. An in vivo assay is described which is designed to test whether 16S rRNA of a translating ribosome can interact with the crb sequence in mRNA in an inducer-dependent reaction. The assay compares the growth rate of cells expressing crb-86 with the growth rate of cells lacking crb-86 in the presence of subinhibitory levels of inducers of cat-86, chloramphenicol, fluorothiamphenicol, amicetin, or erythromycin. Under these conditions, crb-86 retarded growth. Deletion of the crb-86 sequence, insertion of ochre mutations into crb-86, or synonymous codon changes in crb-86 that decreased its complementarity with 16S rRNA all eliminated from detection inducer-dependent growth retardation. Lincomycin, a ribosomally targeted antibiotic that is not an inducer of cat-86, failed to selectively retard the growth of cells expressing crb-86. We suggest that cat-86 inducers enable the crb-86 sequence in mRNA to base pair with 16S rRNA of translating ribosome. When the base pairing is extensive, as with crb-86, ribosomes become transiently trapped on crb and are temporarily withdrawn from protein synthesis to the extent that growth rate declines. Site-specific positioning of an antibiotic-stalled ribosome is a hallmark of the translational attenuation model. The proposed rRNA-mRNA interaction may precisely position the ribosome on the stall site and perhaps contributes to stabilizing the ribosome leader mRNA complex.
authors.affiliation: Department of Biological Sciences, University of Maryland Baltimore County, Catonsville 21228.
authors.forename: E J
authors.initials: EJ
authors.lastname: Rogers
authors.forename: N P
authors.initials: NP
authors.lastname: Ambulos
authors.forename: P S
authors.initials: PS
authors.lastname: Lovett
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics* / Q000254:growth & development; D001483:Base Sequence; D002701:Chloramphenicol / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002872:Chromosome Deletion; D005798:Genes, Bacterial / Q000187:drug effects; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D015183:Restriction Mapping; D012270:Ribosomes / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D021382:Protein Sorting Signals; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D002701:Chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase