The control region for erythromycin resistance: free energy changes related to induction and mutation to constitutive expression.

Abstract

The mRNA sequence in plasmid pE194 encoding the gene responsible for erythromycin resistance has been deduced from the corresponding DNA sequence. The putative control region of this gene located at the 5′ end of the messenger transcribed along with the coding sequence comprises a series of inverted complementary repeat sequences that can redistribute and assume alternative double stranded conformations. Activation of the messenger depends on the degree to which ribosomes inhibited (“stalled”) by erythromycin disrupt the secondary structure in a part of the control region that simultaneously codes for a small peptide and overlaps two of the inverted repeat sequences. In this model, ribosome stall is produced by erythromycin, the inducer, and the result of inverted repeat sequence redistribution is the unmasking of a ribosome binding site for synthesis of the protein that mediates the resistance phenotype. Free energy calculations have been made for the postulated “active” and “inactive” conformations of the control region as well as for 4 classes of mutants that have been mapped in the control region. In addition, calculations have been made for hypothetical sequence alterations corresponding to mutants not yet found. Two modes of induction have been defined and their associated free energy changes calculated; these are (i) preemptive induction, in which ribosome stall resulting in disruption of stem sequence conformation causes one inverted repeat sequence to preempt a second. In the resultant redistribution of complementary inverted repeat sequences, the control region assumes the “active” conformation owing to unmasking of a sequestered ribosome binding site, and (ii) direct dissociation of the critical stem sequences to unmask the sequenstered ribosome binding site, a reaction that bypasses the preemptive association mechanism. The base change found in one of the mutants is consistent with the preemptive mode of induction according to which ribosome stall to an extent that destabilizes the firt three of 13 possible paired bases in the inactive form of the messenger suffices to induce expression of the resistance phenotype. The single-base-change mutations to constitutive expression most commonly found are precisely those that produce the greatest loss of negative free energy; this majority group affects the control region sequence further downstream and appears to act by the direct dissociation mode. Finally, a pair of weakly interacting inverted repeat sequences flanking the more strongly interacting repeats is present; it is postulated that this outer pair serves during the initial phase of induction, as well as later, to turn off expression of the induced messenger after removal of an inductive stimulus. The wide variety of possible mutations capable of disrupting stability of the control region suggests a model to explain the diversity of “partial constitutive” phenotypes found in earlier studies.