Life without tRNAArg–adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes

Abstract

In most bacteria, two tRNAs decode the four arginine CGN codons. One tRNA harboring a wobble inosine (tRNAArgICG) reads the CGU, CGC and CGA codons, whereas a second tRNA harboring a wobble cytidine (tRNAArgCCG) reads the remaining CGG codon. The reduced genomes of Mycoplasmas and other Mollicutes lack the gene encoding tRNAArgCCG. This raises the question of how these organisms decode CGG codons. Examination of 36 Mollicute genomes for genes encoding tRNAArg and the TadA enzyme, responsible for wobble inosine formation, suggested an evolutionary scenario where tadA gene mutations first occurred. This allowed the temporary accumulation of non-deaminated tRNAArgACG, capable of reading all CGN codons. This hypothesis was verified in Mycoplasma capricolum, which contains a small fraction of tRNAArgACG with a non-deaminated wobble adenosine. Subsets of Mollicutes continued to evolve by losing both the mutated tRNAArgCCG and tadA, and then acquired a new tRNAArgUCG. This permitted further tRNAArgACG mutations with tRNAArgGCG or its disappearance, leaving a single tRNAArgUCG to decode the four CGN codons. The key point of our model is that the A-to-I deamination activity had to be controlled before the loss of the tadA gene, allowing the stepwise evolution of Mollicutes toward an alternative decoding strategy.

Transfer RNA Misidentification Scrambles Sense Codon Recoding

“…[11] Only a small fraction of M. capricolum tRNA Arg ACG does not undergo an adenosine (A)-to-inosine (I) editing event in the anticodon. [12] Whereas edited tRNA Arg ICG recognizes CGA, CGU, CGC but not CGG, unmodified tRNA Arg ACG can weakly decode CGG. Mycoplasma species are ideal organisms for synthetic biology studies due to their small genome size.…”

RNA editing in bacteria recodes multiple proteins and regulates an evolutionarily conserved toxin-antitoxin system

“…Future studies are needed to examine if all detected mRNA editing events have functional consequences or whether some of them represent an "accidental" activity due to sequence/structure similarity to tadA's substrates. tadA is found in most bacterial species (Yokobori et al 2013). Therefore, our work sets the stage for investigating RNA editing in other bacterial species that harbor this enzyme.…”

Experimental challenges of sense codon reassignment: An innovative approach to genetic code expansion

“…The CGG codon usage varies greatly among those mycoplasmas that have lost the tadA gene and those that retain it. The absence (or dispensability) or reduced activity of TadA suggests that the organism is evolving towards a reduced genome and unconventional decoding approaches [35,56]. In M. capricolum , the inefficient expression of tadA , either due to lowered expression or inefficient catalytic activity of the enzyme, would allow the presence of low amounts of that are not deaminated, and this could function in translating all of the CGN codons, including CGG.…”

“…Mycoplasmas also have a non-standard genetic code and a reduced number of tRNAs (ranging from [29][30][31][32][33][34][35], where UGA is read as tryptophan. Many mycoplasma species are also missing several cognate tRNAs, the most obvious being some of tRNA Arg isotypes [29][30][31].…”

“…This leaves the codon CGG ''open'', due to lack of the cognate tRNA CCG . The mycoplasma species that have only the two tRNA Arg species also use the CGG codons very sparingly [35]. Towards achieving the first experimental study of sense codon recoding, we attempted to reassign the CGG codon and evaluate feasibility of other rare sense codons in the mycoplasma genome.…”