8.3 Microbiology - Increased Translation Error Rates and Long-term Survival
Abstract
Quality control of accurate protein translation is governed by the specificity of aminoacyl-tRNA synthetases (aaRS), enzymes which are responsible for correct charging of tRNAs with their cognate amino acid. Loss of this quality control either through misincorporation or defective editing will result in ÒmistranslationÓ leading to a loss of protein structural and functional integrity. While mistranslation is normally rare, it has been recently observed that E. coli cells that lack the quality control function of an aaRS have a competitive advantage over wild-type cells under long-term survival conditions and that these quality control mutants have a higher mutation rate. To further probe this seemingly paradoxical phenomenon, this proposal seeks to determine how translation errors contribute to survival, whether translation errors directly influence the mutation rate and to assess the relative benefit of different translation errors. It is hypothesized that elevated translation errors improve survival during long-term growth (where the growth rate is considerably slowed relative to exponential phase) by increased translational capacity in the face of dwindling resources and/or by translation of a more diverse proteome. In editing-defective E. coli cells (containing a mutant PheRS leading to misincorporation of Tyr for Phe) grown under long term conditions, altered increased translation capacity will be assessed by the loss of metabolic and transcriptomic markers characteristic of the stringent response (which normally shuts down the translational machinery during amino acid starvation) and by polysome profiling which provides an indication of active 70S ribosomes associated with mRNA. To assess the presence of a more diverse proteome, mass spectrometry-based assays will be used to quantify the mistranslation level of targeted proteins known to have central roles in translation. To establish a possible link between mistranslation and higher mutation rates, editing defective cells will be assessed for an abundance of the GASP phenotype by competing aged versus younger cells grown through stationary phase. Overall mutational frequency and the mutational spectrum of editing-defective cells exhibiting a GASP phenotype will be assessed by genomic sequencing and compared against the sequence of a wild-type strain exhibiting the GASP phenotype. To determine if different translation errors are equally beneficial, a second editing-defective strain will be constructed (mutant of IleRS which should result in misincorporation of either Val or Leu for Ile) and assessed for its ability to compete with wild-type under long-term stationary phase. Overall mutational frequency will be determined and effects of a double mutant (editing defective alleles of IleRS and PheRS) will be assessed.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 12, 2017
- Source ID
- W911NF1510105
Entities
People
- Beth Lazazzera
Organizations
- Army Contracting Command
- United States Army
- University of California, Los Angeles