Chloramphenicol-induced translation of cat-86 mRNA requires two cis-acting regulatory regions

Abstract

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.

Drug-free induction of a chloramphenicol acetyltransferase gene in Bacillus subtilis by stalling ribosomes in a regulatory leader

“…It was suggested that chloramphenicol modifies ribosomal function, permitting ribosomes to destabilize a stem-loop structure in cat mRNA which sequesters the cat ribosome-binding site (RBS) (13,19). Evidence that is consistent with this posttranscriptional regulatory model has been provided for the cat-86 gene, as well as for inducible cat genes resident on the Staphylococcus plasmids pC194 and pUB112 (1,2,4,7,8,11,12,19,22,27). However, despite efforts in several laboratories, there is no experimental evidence to support models proposed to explain how a ribosome might disrupt a cat RNA stem-loop structure (7,8,19).…”

Isolation and characterization of chromosomal promoters of Streptococcus salivarius subsp. thermophilus

“…(B) Determination of promoter strength by RNA dot blot analysis. Different concentrations of RNA (10 or 5 p.g) from S. thermophilus A054 containing either pTG244 or recombinant plasmid pTG244-3, pTG244-8, pTG244-14, pTG244-15, pTG244-20, or pTG244-25 were hybridized with the oligonucleotide probe TG1769 (5'-GTGAAAGT GCTCTTTTCGCAG-3') specific for the cat-86 transcript (positions +48 to +28 of the cat-86 sequence [1]). the nucleotide sequence of the putative promoter P25 revealed the presence of consensus sequences at the 3' end of the fragment but no flanking ORF.…”

Improved vector for promoter screening in lactococci

“…Plasmid pPL703 AAC1 (4,967 bp) was the source of the constitutively expressible cat-86 gene. This plasmid carris a deletion and duplication within the region involved in the control of cat-86 gene inducibility, making it constitutively expressible (2). Plasmids pBV502 and pPL703AAC1 were digested with restriction enzymes PstI and HindIII.…”

“…Studies of cat-86 gene expression in B. subtilis have shown that inducibility results from the activation of the translation of cat-86 mRNA by drug-modified ribosomes in a process that has been termed translational attenuation (1). The constitutively expression version of the cat-86 gene in B. subtilis plasmid pPL708AAC1 specifies chloramphenicol acetyltransferase (CAT) activity that is four times higher than the activity specified by the inducible version of the gene behind the same promoter (2).…”