Functional annotation of class I lysyl-tRNA synthetase phylogeny indicates a limited role for gene transfer

Abstract

Functional and comparative genomic studies have previously shown that the essential protein lysyl-tRNA synthetase (LysRS) exists in two unrelated forms. Most prokaryotes and all eukaryotes contain a class II LysRS, whereas most archaea and a few bacteria contain a less common class I LysRS. In bacteria the class I LysRS is only found in the alpha-proteobacteria and a scattering of other groups, including the spirochetes, while the class I protein is by far the most common form of LysRS in archaea. To investigate this unusual distribution we functionally annotated a representative phylogenetic sampling of LysRS proteins. Class I LysRS proteins from a variety of bacteria and archaea were characterized in vitro by their ability to recognize Escherichia coli tRNA(Lys) anticodon mutants. Class I LysRS proteins were found to fall into two distinct groups, those that preferentially recognize the third anticodon nucleotide of tRNA(Lys) (U36) and those that recognize both the second and third positions (U35 and U36). Strong recognition of U35 and U36 was confined to the pyrococcus-spirochete grouping within the archaeal branch of the class I LysRS phylogenetic tree, while U36 recognition was seen in other archaea and an example from the alpha-proteobacteria. Together with the corresponding phylogenetic relationships, these results suggest that despite its comparative rarity the distribution of class I LysRS conforms to the canonical archaeal-bacterial division. The only exception, suggested from both functional and phylogenetic data, appears to be the horizontal transfer of class I LysRS from a pyrococcal progenitor to a limited number of bacteria.

FIG. 1.

Secondary structure of undermodified E. coli tRNA^Lys. Abbreviations: D, dihydrouridine; ψ, pseudouridine; m⁷G, 7-methylguanosine; X, putative 3-(3-amino-3-carboxypropyl) uridine modification. The undermodified variants used in this study contain uridine in place of 5-methylaminomethyl-2-thiouridine at position 34 and adenosine in place of N⁶-threonylcarbamoyladenosine at position 37. Changes in the anticodon nucleotides (nucleotides 34 to 36) are indicated.

FIG. 2.

Aminoacylation of E. coli tRNA^Lys variants by class I His₆-LysRS proteins. Aminoacylation reactions were performed as described in the text (20-μl samples) in the presence of 4 μM tRNA and 100 μM [¹⁴C]lysine. Comparison of charging of wild-type and mutant tRNA^Lys using class I LysRS enzymes from S. aromaticivorans (200 nm of enzyme) (A and B) and F. acidarmanus (2 μM enzyme) (C and D).

FIG. 3.

Aminoacylation of E. coli tRNA^Lys variants by M. barkeri class II His₆-LysRS. Aminoacylation reactions were performed as described in the text (20-μl samples) in the presence of 4 μM tRNA, 100 μM [¹⁴C]lysine, and 2 μM enzyme. (A) Position 34 variants; (B) position 35 and 36 variants.

FIG. 4.

Maximum-likelihood phylogenetic tree of class I LysRS sequences. The tree has been rooted using GluRS sequences. Numbers indicate the percentage occurrence of nodes after 10,000 puzzle steps.

FIG. 5.

Alignment of tRNA^Lys sequence (UUU anticodon) from organisms encoding class I LysRSs. Nucleotide 26 is indicated and shown in boldface type, and the anticodon sequence is underlined. Abbreviations: Rpa, Rhodopseudomonas palustris; CC, Caulobacter crescentus; MMa, M. magnetotacticum; RP, R. prowazekii; Wol, Wolbachia sp.; SA, S. aromaticivorans; ML, Mesorhizobium loti; RS, Rhodobacter sphaeroides; PF, Pyrococcus furiosus; PA, Pyrococcus abysii; PH, Pyrococcus horikoshii; BB, B. burgdorferi; SC, Streptomyces coelicolor; TD, Treponema denticola; TP, Treponema pallidum; HB, Halobacterium sp.; AP, Aeropyrum pernix; MT, M. thermoautotrophicum; MJ, M. jannaschii; MM, M. maripaludis; AF, Archaeoglobus fulgidus; MB, M. barkeri; FA, F. acidarmanus; TV, Thermoplasma volcanium; TA, Thermoplasma acidophilum. Sequences of genes encoding tRNA^Lys (UUU) were obtained from The Institute for Genomic Research (http://www.tigr.org), the Sanger Center (http://www.sanger.ac.uk/Projects/), and public databases (http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi).