Protection of tRNA^Asp amino acid accepting capacity by E.coli AspRS or YadB catalyzed aminoacylation prior to periodate oxidation. (A) Extent of tRNA^Asp aspartylation by AspRS (gray bars) and glutamylation by YadB (white bars). (B) The same experiment with tRNA^Asp lacking its single stranded 3′ terminus. (C) Same experiments with tRNA^Asp treated with periodate.

Sequence comparison of tRNA^Glu acceptor arm and tRNA^Asp anticodon stem and loop from bacteria encoding YadB and expansion of the comparison to tRNA^Asn, tRNA^His and tRNA^Tyr anticodon stems and loops. tRNAs from 17 bacterial species were compared. (A) Escherichia coli tRNA^Asp and tRNA^Glu are presented in a 2-dimensional projection of their L-shaped architecture. Nucleotides targeted by the comparison are displayed and the amino acid-accepting nucleotides (Q34 and A76) indicated by an arrow. Numbering of nucleotides is according to Sprinzl et al. (32). For each bacterial species tRNA^Asp anticodon stem and loop positions (base pairs 27–43 to 30–40 and nucleotides 39 and 38) were compared to the corresponding positions of the tRNA^Glu acceptor stem (base pairs 4–69 to 1–72 and nucleotides 73 and 74) from the same species. When nucleotides are the same as in tRNA^Asp and tRNA^Glu from E.coli tRNAs, they are displayed in white on a black box. For clarity, only nucleotides 43–38 from tRNA^Asp and 69–74 from tRNA^Glu are displayed. Consensus sequences of the 38–43 stretches in tRNA^Asp and 74–68 stretches in tRNA^Glu are given together with the consensus of the complementary residues (30–27 and 1–4 stretches). Residues in italic are partially conserved; R stands for purine and Y for pyrimidine. The sequences displayed are ranked according to the YadB classification (14). (B) 2-Dimensional representation of E.coli tRNA^Asp and the consensus sequences in the anticodon branch of the 17 bacterial species. When a nucleotide is the same as its counterpart from the consensus sequence of tRNA^Glu it is displayed in white on a black box.

Analysis of the amino acid accepting capacity of E.coli tRNA^Asp when aminoacylated by AspRS, YadB and by both. (A) Extents of pure tRNA^Asp charging with E.coli AspRS (gray bar), YadB (white bar) or AspRS and YadB (black bar). (B) The amino acids acylating tRNA^Asp are identified by TLC as described (10).

Amino acid accepting capacities of E.coli native tRNA^AspQ34 and queuosine-lacking tRNA^AspG34. (A) Kinetics of aminoacylation of tRNA^AspQ34 (top) and tRNA^AspG34 (bottom) by E.coli AspRS (black triangles) and YadB (black squares). (Insets) 2-Dimensional TLC analysis of the 5′ phosphate nucleosides present in the two tRNAs (the arrows indicate the positions where pQ migrates). The structures of Q and G are shown at the left with the arrows indicating the OH groups of the cyclopentenyldiol ring that are the putative Glu acceptors on Q. (B) Lineweaver–Burk plots allowing determinations of the K_m (open triangles) of YadB for native tRNA^Asp and K_i (open squares) of tRNA^AspQ34 for aminoacylation of native tRNA^Asp by YadB.

A tentative model of the complex of E.coli tRNA^Asp with cognate YadB. (A) The YadB molecular surface is displayed in dark blue, the loop in lighter blue and adenylate in sphere representation. The docking procedure was based on the constraint that the reactive group, the cyclopentenyldiol ring of Q34, like the ribose from A76, should be close to the Glu–adenylate phosphate–Glu scissile bond. The Q34 cyclopentenyldiol ring was therefore used as a pivot around which the tRNA rigid body moved. The tRNA^Asp is displayed as an atom color coded molecular surface. (Inset) Close-up of the interaction illustrating the vicinity of the adenylate donor molecule and the acceptor group, Q34 of tRNA^Asp. Adenylate and the tRNA^Asp domain with Q base are displayed in stick representation. (B) Rotation of ∼120° around a vertical axis and ∼30° clockwise in the plane.