The influence of identity elements on the aminoacylation of tRNA^Arg by plant and Escherichia coli arginyl‐tRNA synthetases

Abstract

Identity elements determine the accurate recognition between tRNAs and aminoacyl-tRNA synthetases. The arginine system from yeast and Escherichia coli has been studied extensively in the past. However, information about the enzymes from higher eukaryotes is limited and plant aminoacyltRNA synthetases have been largely ignored in this respect. We have designed in vitro tRNA transcripts, based on the soybean tRNA Arg primary structure, aiming to investigate its specific aminoacylation by two recombinant plant arginyl-tRNA synthetases and to compare this with the enzyme from E. coli. Identity elements at positions 20 and 35 in plants parallel those previously established for bacteria. Cryptic identity elements in the plant system that are not revealed within a tRNA Arg consensus sequence compiled from isodecoders corresponding to nine distinct cytoplasmic, mitochondrial and plastid isoaccepting sequences are located in the acceptor stem. Additionally, it has been shown that U20a and A38 are essential for a fully efficient cognate E. coli arginylation, whereas, for the plant arginyl-tRNA synthetases, these bases can be replaced by G20a and C38 with full retention of activity. G10, a constituent of the 10 : 25 : 45 tertiary interaction, is essential for both plant and E. coli activity. Amino acid recognition in terms of discriminating between arginine and canavanine by the arginyl-tRNA synthetase from both kingdoms may be manipulated by changes at different sites within the tRNA structure.

Expression of genes for selected plant aminoacyl-tRNA synthetases in the abiotic stress

“…Research on plant aaRSs revealed their cellular localization (Rokov-Plavec et al 2008, Duchêne et al 2009, Kekez et al 2016, substrate specificity (Rokov et al 2008, Rokov and Weygand-Durasevic 2009, Rokov-Plavec et al 2002, 2004, Aldinger et al 2012, fidelity (Rokov-Plavec et al 2013, Lee et al 2016, Hoffman et al 2019 and protein interactors (Yang et al 2018, Kekez et al 2019. Thus far, only one crystal structure of plant aaRS is reported (Kekez et al 2019).…”

Molecular evidence for the evolution of the eukaryotic mitochondrial arginyl‐tRNA synthetase from the prokaryotic suborder Cystobacterineae

“…In the case of arginyl‐tRNA synthetase, the enzyme from Escherichia coli requires nucleotide A20 as a major identity element in tRNA and is unable to arginylate the corresponding yeast tRNA that possesses C20 , although the yeast enzyme recognizes the bacterial tRNA. Extrapolating this observation to the Cyto / Mito system, it is well‐established that the Cyto arginyl‐tRNA synthetase in mammals and plants relies on the presence of A20 . Conversely, Coleopteran Mito tRNA Arg lacks a canonical A20 base and is not aminoacylated by the (Cyto‐like) E. coli enzyme .…”

Identity elements for the aminoacylation of metazoan mitochondrial tRNAArghave been widely conserved throughout evolution and ensure the fidelity of the AGR codon reassignment

“…However, neither was a substrate for a higher eukaryotic (plant) cytoplasmic arginyl-tRNA synthetase (data not shown) that requires an A20 identity element. 43 Parenthetically, evidence has been presented that the yeast cytoplasmic arginyltRNA synthetase has mitochondrial origins. 53,54 Finally, in a third class of sponges, the Calcarea, the CGN arginine codon has been reassigned to glycine, retaining tRNA Arg UCU as the sole arginine decoder.…”

“…The identity elements are independent of the sequence of the distal acceptor stem. The major identity element A20, that has been described for the E.coli, 44 mammalian cytoplasmic 45 and plant arginyltRNA synthetases, 43 has been lost in insect mitochondria during the evolution of highly variable and, in some species, rudimentary D-stem/loops. The activity of the C. duodecimpunctata tRNA Ala whose anticodon had been replaced by UCG supports the conclusion that the discriminator base U 73 (tRNA Arg ) or A 73 (tRNA Ala ) does not play a role in recognition.…”

“…A major identity element for the recognition of the C. brasiliensis mitochondrial tRNA Arg by its cognate synthetase resides, as is the case for cytoplasmic tRNA Arg from several organisms, 43 is the central anticodon position, C35. Replacement by G35 in the cognate tRNA eliminated acceptor activity.…”