Posttranscriptional modification of mRNA conformation: Mechanism that regulates erythromycin-induced resistance

The nucleotide sequence of a gene in plasmid pE194 responsible for erythromycin-induced resistance, including regulation of the resistance phenotype, is reported. A DNA fragment from plasmid pE194, obtained by digestion with Taq I restriction endonuclease, was cloned in Bacillus subtilis by using pC194 as the plasmid cloning vector. Erythromycin-resistant, inducible transformant clones containing the Taq I fragment A were obtained in which the expression of resistance was similar to that found in the original pE194 background; an interpretative model of the regulation of the erythromycin-resistance determinant is proposed based on the sequence of the Taq I A fragment. The cloned Taq I A fragment consists of 1442 base pairs and has open reading frames capable of coding for a peptide and a protein containing 19 and 243 amino acids, respectively, referred to as the "leader peptide" and "29,000 protein." Between the putative transcriptional start site and the ribosome binding site for 29,000-protein synthesis, the promoter region contains four complementary inverted repeat sequences named "1, 2, 3, and 4," respectively, in which 1 is complementary to 2, 2 is complementary to 3, and 3 is complementary to 4. Sequence 1 encodes the COOH-terminal half of the leader peptide, whereas the ribosome binding site for synthesis of 29,000 protein is sequestered in a loop formed by the association of 3 and 4. The 29,000-protein promoter region does not appear to contain any transcription stop signal. We propose a model for regulation of erythromycin resistance according to which ribosomes engaged in leader peptide synthesis are partially inhibited by optimal inducing (i.e., subinhibitory) concentrations of erythromycin that, in turn, cause an accumulation of these partially inhibited ("stalled") ribosomes in sequence 1. During induction, the translationally inactive states of association of the inverted repeats, postulated to be 1 plus 2 and 3 plus 4, respectively, are perturbed by a high level of stalled ribosome occupancy in sequence 1, and in the resultant redistribution, 2 associates with 3, freeing 4 and thereby freeing the ribosome binding site sequestered by the association of 3 and 4. Sequence alterations at the 5' end of the 29,000-protein coding region associated with mutation to constitutive expression have been localized to the inverted complementary repeats, and determination of base changes in eight mutants are all capable of reducing the stability of the postulated stems in a manner consistent with predictions made by the model.