Effect of amber suppressor mutation on the frequency of mutants and evolution of Escherichia coli

• Grant number: 20/15873-3
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: September 01, 2021
• End date: May 31, 2025
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Beny Spira
• Grantee: Gabriella Trombini Machado
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

A mutation that restores, partially or totally, the loss of function caused by another mutation is called "suppressor". One of the most classic cases of suppressor mutations refers to the omission of stop codons by suppressor tRNAs. These species of tRNAs carry nucleotide substitutions in their anticodons that start to recognize one of the three stop codons. In this way, strains that carry suppressive mutations tend toform proteins longer than the original sequence and may have their function partially impaired. Previous experiments in our laboratory showed that PHO constitutive mutants derivatives of Escherichia coli K-10 have a higher frequency of mutants compared to the MG1655 strain. The K-10 strain has a suppressor mutation in tRNAGln that allows it to insert the amino acid glutamine in response to the amber stop códon (UAG). When analyzing Escherichia coli genes that end with TAG, we find mutH, mutT and mutY, which encode proteins involved in the DNA repair mechanism. Therefore, we hypothesized that the highest frequency of mutants in the K-10 strain is due to the presence of partially defective MutH, MutT and MutY repair proteins, as they were stretched due to the suppression of the amber stop codon. To test this hypothesis, point mutations will be introduced in the tRNAGln gene and in each of the mutH, mutT and mutY genes. Experiments to measure mutant frequency and mutation rate will be performed for each of these mutants separately and also for double and triple mutants of the mutH, mutT and mutY genes. A second hypothesis related to the existence of amber suppressor mutations is that genes have evolved to minimize the suppression effects favoring the presence of other stop codons (UGA or UAA) subsequent to UAG codons in the same reading frame. This "second stop codon" would actually finish translating these proteins. Thus, the more essential the gene is for the bacterium, the shorter the distance between the UAG codon and the "second stop codon". This assumption will be evaluated in silico. (AU)