Abstract
A fundamental feature of life is that ribosomes read the genetic code in messenger RNA (mRNA) as triplets of nucleotides in a single reading frame. Mutations that shift the reading frame generally cause gene inactivation and in essential genes cause loss of viability. Here we report and characterize a +1-nt frameshift mutation, centrally located in rpoB, an essential gene encoding the beta-subunit of RNA polymerase. Mutant Escherichia coli carrying this mutation are viable and highly resistant to rifampicin. Genetic and proteomic experiments reveal a very high rate (5%) of spontaneous frameshift suppression occurring on a heptanucleotide sequence downstream of the mutation. Production of active protein is stimulated to 61-71% of wild-type level by a feedback mechanism increasing translation initiation. The phenomenon described here could have broad significance for predictions of phenotype from genotype. Several frameshift mutations have been reported in rpoB in rifampicin-resistant clinical isolates of Mycobacterium tuberculosis (Mtb). These mutations have never been experimentally validated, and no mechanisms of action have been proposed. This work shows that frameshift mutations in rpoB can be a mutational mechanism generating antibiotic resistance. Our analysis further suggests that genetic elements supporting productive frameshifting could rapidly evolve de novo, even in essential genes.
Highlights
| | | | rpoB frameshift suppression antibiotic resistance evolution gene regulation, that the mutants carry frameshift suppressor mutations [15,16,17], or that the messenger RNA (mRNA) contains sequence elements that promote a high level of ribosomal shifting into the correct reading frame to support cell viability [10]
Ribosomal frameshifting errors occur rarely, but when they do the consequences for protein function are generally catastrophic, and frameshift mutations are generally only observed in nonessential genes
In this context we were intrigued by reports of clinical isolates of Mycobacterium tuberculosis (Mtb) carrying frameshift mutations [11,12,13,14], or nonsense mutations [12, 29,30,31], in the essential gene rpoB
Summary
| | | | rpoB frameshift suppression antibiotic resistance evolution gene regulation (unlikely if the DNA sequence analysis was done properly), that the mutants carry frameshift suppressor mutations [15,16,17], or that the mRNA contains sequence elements that promote a high level of ribosomal shifting into the correct reading frame to support cell viability [10]. Interest in understanding these mutations goes beyond Mtb and concerns more generally the potential for rescue of mutants that acquire a frameshift mutation in any essential gene We addressed this by isolating a mutant of Escherichia coli carrying a frameshift mutation in rpoB and experimentally dissecting its genotype and phenotypes
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