N P Ambulos

A transciption termination signal immediately precedes the coding sequence for the chloramphenicol-inducible plasmid gene cat-86

SummaryThe plasmid gene cat-86 specifies chloramphenicol-inducible, chloramphenicol acetyltransferase in Bacillus subtilis. The inducible regulation is independent of the promoter that is used to activate cat-86 and is independent of the cat-86 coding sequence. We have proposed that the regulation of cat-86 results from the transcription of a pair of inverted-repeat sequences that immediately precede the coding sequence. These transcripts are predicted to sequester the cat-86 ribosome binding site in a stable RNA stemloop which, in theory, should block the ribosome binding site from pairing with 16S rRNA. Inducible expression of cat-86 may therefore result in part from regulation of the translation of cat-86 mRNA. However, chloramphenicol-induction correlates with increased levels of cat-86 mRNA and the RNA stem-loop preceding the cat-86 coding sequence structurally resembles a rho-independent transcription terminator. We have therefore tested the inverted-repeats as a potential site of transcription termination. Transcription studies performed in vitro using SP6 RNA polymerase and in vivo by S1 mapping demonstrate that a substantial fraction of the potential cat-86 transcripts terminate at a site immediately 3′ to the inverted-repeats. The results of the in vivo experiments suggest that the termination signal may be partially relieved by growth of cells in chloramphenicol.

Induction of CAT mRNA Translation by Chloramphenicol: an Example of Translational Attenuation

The role of crb-86 in cat-86 induction may be a function of both its complementarity with 16S rRNA and the crb -@#@ encoded peptide. Solving the role of crb. as a gene regulatory device will dissect ribosome function in a manner not possible by conventional methodology much in the same vein as other recently discovered variations of ribosome function (12).