A truncated aminoacyl-tRNA synthetase modifies RNA

Abstract

Aminoacyl-tRNA synthetases are modular enzymes composed of a central active site domain to which additional functional domains were appended in the course of evolution. Analysis of bacterial genome sequences revealed the presence of many shorter aminoacyl-tRNA synthetase paralogs. Here we report the characterization of a well conserved glutamyl-tRNA synthetase (GluRS) paralog (YadB in Escherichia coli) that is present in the genomes of >40 species of proteobacteria, cyanobacteria, and actinobacteria. The E. coli yadB gene encodes a truncated GluRS that lacks the C-terminal third of the protein and, consequently, the anticodon binding domain. Generation of a yadB disruption showed the gene to be dispensable for E. coli growth in rich and minimal media. Unlike GluRS, the YadB protein was able to activate glutamate in presence of ATP in a tRNA-independent fashion and to transfer glutamate onto tRNA(Asp). Neither tRNA(Glu) nor tRNA(Gln) were substrates. In contrast to canonical aminoacyl-tRNA, glutamate was not esterified to the 3'-terminal adenosine of tRNA(Asp). Instead, it was attached to the 2-amino-5-(4,5-dihydroxy-2-cyclopenten-1-yl) moiety of queuosine, the modified nucleoside occupying the first anticodon position of tRNA(Asp). Glutamyl-queuosine, like canonical Glu-tRNA, was hydrolyzed by mild alkaline treatment. Analysis of tRNA isolated under acidic conditions showed that this novel modification is present in normal E. coli tRNA; presumably it previously escaped detection as the standard conditions of tRNA isolation include an alkaline deacylation step that also causes hydrolysis of glutamyl-queuosine. Thus, this aminoacyl-tRNA synthetase fragment contributes to standard nucleotide modification of tRNA.

Fig. 1.

Growth of the W3110ΔyadB strain. Growth at 37°C of E. coli strain W3110 and two independent isolates of W3110ΔyadB on LB and M9 plates.

Fig. 2.

Glutamate activation and transfer onto tRNA by YadB. (A) YadB-dependent amino acid activation measured by ATP-PP_i exchange. The lanes are: -YadB; -ATP; -Glu; -tRNA, Glu in the absence of tRNA; +tRNA, Glu in the presence of tRNA; +Asp, Asp alone; +19 AA, all amino acids except Glu. (B) Charging of tRNA fractions from reverse-phase HPLC with aspartate using E. coli AspRS (dotted line) and with glutamate using YadB (solid line). (C) Charging of purified tRNA^Asp with aspartate using E. coli AspRS (□) and with glutamate using YadB (○).

Fig. 3.

Analysis of periodate-treated Glu–tRNA^Asp and Asp–tRNA^Asp. (A) Glu–tRNA^Asp and Asp–tRNA^Asp was treated with periodate and then lysine, deacylated, and subjected to gel electrophoresis for size determination. tRNA was visualized by hybridization with ³²P-labeled probes against tRNA^Asp. N, position of intact tRNA^Asp; N-1, tRNA^Asp without the terminal adenosine; N^*, YadB-generated Glu–tRNA^Asp lacking the terminal adenosine; tRNA control. (B) Glu–tRNA^Asp was treated with periodate, deacylated and then recharged with either glutamate and YadB (○) or aspartate and E. coli AspRS (•). Untreated tRNA is aminoacylated by YadB (□) and AspRS (▪).

Fig. 4.

Simultaneous acylation of purified E. coli tRNA^Asp with aspartate and glutamate. Charging with [¹⁴C]Asp and AspRS (□), [¹⁴C]Glu using YadB (○), and [¹⁴C]Asp and AspRS in the presence of [¹⁴C]Glu and YadB (•). Accounting for the different specific activities of Asp and Glu (see Materials and Methods), aminoacylation at the 20 min time point is >90%.

Fig. 5.

Glutamylation and aspartylation of queuosine-deficient tRNA and structures of queuosine and glutamyl–queuosine. (A) tRNA isolated from the queuosine-deficient E. coli JE7350 strain and wild-type tRNA was acylated in vitro. (▪) wild-type tRNA, [¹⁴C]Asp, and AspRS; (•) Q-deficient tRNA, [¹⁴C]Asp, and AspRS; (□) wild-type tRNA, [¹⁴C]Glu, and YadB; (○) Q-deficient tRNA, [¹⁴C]Glu, and YadB. (B) Queuosine and glutamyl–queuosine.

Fig. 6.

Liquid chromatography/electrospray ionization-mass spectrometry analysis of nucleoside mixtures derived from purified W3110ΔyadB tRNA^Asp before and after YadB-catalyzed glutamylation (A–C) and from unfractionated W3110 tRNA isolated under acidic conditions (D and E). Reconstructed ion chromatograms for m/z 539.2, the MH⁺ ion expected for glutamyl–queuosine (A); m/z 410.2, the MH⁺ ion expected for queuosine (B); m/z 539.2 from tRNA^Asp (C). Selected ion recording for m/z 539.2, the MH⁺ ion expected for glutamyl–queuosine (D) and m/z 410.2, the MH⁺ ion expected for queuosine (E). The glutamyl–queuosine peak is marked with an asterisk (A and D). Because this figure contains two different experimental runs (A–C, and D and E), two queuosine standards (B and E) are included.