Prolyl-tRNA synthetase from Thermus thermophilus is eukaryotic-like but aminoacylates prokaryotic tRNAPro

Abstract

Aim. Cloning, sequencing and expression of the gene encoding prolyl-tRNA synthetase, a class IIa enzyme, from the extreme thermophile T. thermophilus HB8 (ProRSTT). Methods. Search for the ProRSTT gene was performed by Southern blot hybridization with chromosomal DNA, the digoxigenin-labeled PCR fragments of DNA being used as a probe. Results. The gene of T. thermophilus HB8 ProRS has been cloned and sequenced. The predicted 477-amino acid protein is significantly more similar in sequence to eukaryotic and archaeal than to eubacterial ProRS. Sequence analysis shows two distinct structural groups of ProRS which most likely had diverged early in evolution: (1) eukaryotic/archaeal group characterized by the absence of an insertion domain between motifs 2 and 3 and by the presence of an extra C-terminal domain beyond the normal class IIa anticodon binding domain; and (2) prokaryotic with a very large insertion between motif 2 and 3 and no extra C-terminal domain. Conclusions. T. thermophilus proS gene was overexpressed in Escherichia coli and overproduced ProRSTT was purified to high homogeneity. In spite of its eukaryotic-like features, T. thermophilus ProRS exhibited highly specific cross-species aminoacylation. The charging ability of the ProRSTT is restricted to prokaryotic tRNAPro

Adaptive evolution: Eukaryotic enzyme's specificity shift to a bacterial substrate

“…Sequence alignment indicates that ProRS shares a higher degree of similarity to enzymes found in the cytoplasm of eukaryotes. For instance, it shares 35% homology with yeast ProRS, 37% with Drosophila melanogaster and human ProRS, contrasting with bacterial ProRSs, where it exhibits only 18% homology with E. coli ProRS and 14% with Chlamydia trachomatis ProRS (Yaremchuk et al, 2012). Phylogenetic analysis, employing full‐length protein sequences alongside sequences from each domain, reveals that TtProRS clusters with other bacterial E‐type ProRSs and plant organellar ProRSs, with the exception of its anticodon binding domain.…”

“…Despite having three tRNA Pro genes, all encode the P‐type tRNA Pro . A prior investigation indicates that T. thermophilus ProRS (TtProRS) selectively charges the P‐type tRNA Pro , yet the precise mechanism remains elusive (Yaremchuk et al, 2012). Our findings reveal that, akin to a P‐type ProRS, TtProRS recognizes the bacterium‐specific acceptor‐stem elements G72/A73, alongside the anticodon elements G35/G36.…”