Visualizing high error levels during gene expression in living bacterial cells

Abstract

To monitor inaccuracy in gene expression in living cells, we designed an experimental system in the bacterium Bacillus subtilis whereby spontaneous errors can be visualized and quantified at a single-cell level. Our strategy was to introduce mutations into a chromosomally encoded gfp allele, such that errors in protein production are reported in real time by the formation of fluorescent GFP molecules. The data reveal that the amount of errors can greatly exceed previous estimates, and that the error rate increases dramatically at lower temperatures and during stationary phase. Furthermore, we demonstrate that when facing an antibiotic threat, an increase in error level is sufficient to allow survival of bacteria carrying a mutated antibiotic-resistance gene. We propose that bacterial gene expression is error prone, frequently yielding protein molecules that differ slightly from the sequence specified by their DNA, thus generating a cellular reservoir of nonidentical protein molecules. This variation may be a key factor in increasing bacterial fitness, expanding the capability of an isogenic population to face environmental challenges.Bacillus subtilis | translational errors | variations in living cells | translation fidelity D NA is duplicated with remarkable fidelity to ensure that accurate genetic information is transmitted from one generation to the next. This information is passed from DNA to RNA and from RNA to protein during gene expression; however, the accuracy of these downstream events is relatively less understood. Although RNA and proteins are generally considered short-lived noninherited molecules, several diseases are now known to arise from errors occurring during transcription and translation (1-3), implying that faithful transfer of genetic information from DNA to proteins is crucial for maintaining proper cellular functions.

Mycobacterial mistranslation is necessary and sufficient for rifampicin phenotypic resistance

“…Typically, the measured error rate for aminoacyl tRNA synthesis using purified enzymes is 1 error per 10,000 reactions. Nonetheless, the very high discrimination of protein translation enzymes as measured in vitro does not explain the relatively high error rates witnessed in vivo (3)(4)(5)(6). These high error rates of up to 10% per codon in both bacteria and eukaryotic cells were tolerated not only in genetically modified mutants (6) but also in WT cells (3,4).…”

“…To investigate this, we measured mistranslation rates in WT mycobacteria grown under different environmental conditions. We used gain of function reporters (3,6,23) that can sensitively detect small increases in the mistranslation rate. The kanamycin kinase (Aph) protein contains a critical aspartate that is necessary to produce resistance to kanamycin (24).…”

“…Nonetheless, the very high discrimination of protein translation enzymes as measured in vitro does not explain the relatively high error rates witnessed in vivo (3)(4)(5)(6). These high error rates of up to 10% per codon in both bacteria and eukaryotic cells were tolerated not only in genetically modified mutants (6) but also in WT cells (3,4). The high error rate was due to lack of discrimination at aminoacyl tRNA formation (4,7) and at the ribosome (3), suggesting that multiple different forms of translational error occur physiologically, especially under stress.…”

“…Error rates exceeding 1/100 amino acid substitutions per codon, such as under stressful conditions (3,4,7), result in a significant proportion of any given translated protein being represented by multiple variants (8), a phenomenon generally thought to be deleterious to the cell. However, these errors result in new variants that could result in new functions within the "statistical proteome" (9, 10), although none have been identified to date.…”

Comment on “Widespread RNA and DNA Sequence Differences in the Human Transcriptome”

“…Because RNA editing and transcriptional noise are known phenomena (2)(3)(4), the novelty of this work resides in the extreme extent to which the differences are reported to happen in human cells. However, although high-throughput sequencing (HTS) does provide unprecedented opportunities to study the transcriptome, Li et al did not properly control for a number of technical limitations of HTS and the downstream analysis of the data, resulting in an unacceptably high false-positive rate within this study.…”