Potential role of phenotypic mutations in the evolution of protein expression and stability

Abstract

Phenotypic mutations (errors occurring during protein synthesis) are orders of magnitude more frequent than genetic mutations. Consequently, the sequences of individual protein molecules transcribed and translated from the same gene can differ. To test the effects of such mutations, we established a bacterial system in which an antibiotic resistance gene (TEM-1 ␤-lactamase) was transcribed by either a high-fidelity RNA polymerase or its error-prone mutant. This setup enabled the analysis of individual mRNA transcripts that were synthesized under normal or error-prone conditions. We found that an increase of Ϸ20-fold in the frequency of transcription errors promoted the evolution of higher TEM-1 expression levels and of more stable enzyme variants. The stabilized variants exhibited a distinct advantage under error-prone transcription, although under normal transcription they conferred resistance similar to wild-type TEM-1. They did so, primarily, by increasing TEM-1's tolerance to destabilizing deleterious mutations that arise from transcriptional errors. The stabilized TEM-1 variants also showed increased tolerance to genetic mutations. Thus, although phenotypic mutations are not individually subjected to inheritance and natural selection, as are genetic mutations, they collectively exert a direct and immediate effect on protein fitness. They may therefore play a role in shaping protein traits such as expression levels, stability, and tolerance to genetic mutations.protein evolution ͉ protein stability ͉ transcriptional errors ͉ mutational robustness A wealth of error-prevention and correction mechanisms have evolved to lower the mutation rates of DNA, resulting in rates of genomic mutations that are in the order of 5 ϫ 10 Ϫ10 nucleotide exchanges per position per generation (1). In contrast, the mutation rates of mRNA transcription and translation, leading to protein synthesis, are Ϸ5 and 6 orders of magnitude higher respectively (2-5). Because the average length of proteins in prokaryotes is Ϸ350 aa (6), synthesis errors would introduce, on average, a phenotypic mutation in 1 of 10 protein copies. For the largest Escherichia coli ORF (7,200 bases) errors might be found in more than half of the copies (7). In general, the generation of multiple mRNA copies from the same gene ensures a sufficient amount of wild-type mRNA and protein molecules. This may explain why the rates of phenotypic mutations have remained relatively high (8). However, in cases where the number of protein copies in the cell is very low, phenotypic mutations may significantly affect fitness (8). Indeed, Ͼ80% of E. coli genes express fewer than a hundred protein copies, and many are produced at Յ20 copies per cell (9). At high expression rates, the fraction of mutated and misfolded proteins can lead to accelerated aggregation and reduced fitness (10-12). Thus, abnormal proteins resulting from phenotypic mutations can burden cells by reducing the amount of functional protein, wasting metabolic resources, engaging folding and degradation pathwa...

Visualizing high error levels during gene expression in living bacterial cells

“…In more general terms, errors arising spontaneously during gene expression may increase protein variety and thereby enable genetically identical cells to display heterogeneous phenotypes. This phenomenon is likely to contribute to the robustness of unicellular organisms, allowing them to respond to fluctuating environments without changing their genotype (6)(7)(8)(9)(10).…”

Evolutionary paths of least resistance

“…First, in the stringent selection regime, where a high abundance of functional TEM-1 is required for bacterial growth, is the primary fitness cost associated with insufficient abundance of functional TEM-1 or the presence of misfolded TEM-1 or both? The results of Goldsmith and Tawfik (9) suggest that misfolded TEM-1 presents a secondary fitness cost relative to lack of functional TEM-1, but a definite answer to this question remains outstanding. Interestingly, in the relaxed selection regime of Bratulic et al (1), where large quantities of TEM-1 are not required, the predominant fitness cost is likely the presence of misfolded TEM-1.…”

“…Bratulic et al's paper (1) adds to a growing body of evidence demonstrating that phenotypic mutations (i.e., mutations occurring solely in the expressed phenotype) may indirectly influence the evolutionary dynamics of the underlying genotype (2,9,(11)(12)(13)(14). In particular, genetic adaptations can protect organisms from the deleterious consequences of phenotypic mutations (1, 9,12).…”

“…Goldsmith and Tawfik set up a system in which TEM-1 was subjected to elevated transcriptional rather than translational errors. They observed adaptations that lead to increased expression level and, when expression level was held constant, increased thermostability (9). Because the errors were introduced at the transcription stage, there was no opportunity for the TEM-1 gene to reduce the error rate directly, through the Fig.…”

“…In particular, genetic adaptations can protect organisms from the deleterious consequences of phenotypic mutations (1, 9,12). This mechanism in turn opens the possibility that elevated phenotypic mutation rates may be beneficial for further adaptation (11,14).…”