Abstract
Pseudouridines are known to be important for optimal translation. In this study we demonstrate an unexpected link between pseudouridylation of tRNA and mutation frequency in Pseudomonas species. We observed that the lack of pseudouridylation activity of pseudouridine synthases TruA or RluA elevates the mutation frequency in Pseudomonas putida 3 to 5-fold. The absence of TruA but not RluA elevates mutation frequency also in Pseudomonas aeruginosa. Based on the results of genetic studies and analysis of proteome data, the mutagenic effect of the pseudouridylation deficiency cannot be ascribed to the involvement of error-prone DNA polymerases or malfunctioning of DNA repair pathways. In addition, although the deficiency in TruA-dependent pseudouridylation made P. putida cells more sensitive to antimicrobial compounds that may cause oxidative stress and DNA damage, cultivation of bacteria in the presence of reactive oxygen species (ROS)-scavenging compounds did not eliminate the mutator phenotype. Thus, the elevated mutation frequency in the absence of tRNA pseudouridylation could be the result of a more specific response or, alternatively, of a cumulative effect of several small effects disturbing distinct cellular functions, which remain undetected when studied independently. This work suggests that pseudouridines link the translation machinery to mutation frequency.
Highlights
In addition to the four standard nucleotides, tRNA molecules acquire numerous post-transcriptional modifications, representing the most abundantly modified molecules in cell
In the current study we found that most TruA and RluA target tRNAs in P. putida overlap with E. coli TruA and RluA targets (Table S2)
The absence of TruA- or RluA-dependent pseudouridylation led to the mutator phenotype of P. putida (Figure 2A)
Summary
In addition to the four standard nucleotides, tRNA molecules acquire numerous post-transcriptional modifications, representing the most abundantly modified molecules in cell. TRNA structure formation and stabilization [1], translation decoding [2,3,4,5], maintenance of Accepted: 21 December 2020 translation accuracy and processivity [6], as well as regulation of stress responses [7,8,9], the Published: 23 December 2020 physiological role of several tRNA modifications is still enigmatic. Pseudouridines (Ψs) are the most widespread nucleotide modifications. Sequence similarities, pseudouridine synthases are divided into five families. Synthases within one family show high conservation in protein sequences but protein sequence similarity between different families is low or totally absent [10,11]. All known pseudouridine synthases possess several conserved structure motifs, similar tertiary
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