Structure of E. coli Glutaminyl-tRNA Synthetase Complexed with tRNA ^Gln and ATP at 2.8 Å Resolution

Abstract

The crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) complexed with its cognate glutaminyl transfer RNA (tRNA(Gln] and adenosine triphosphate (ATP) has been derived from a 2.8 angstrom resolution electron density map and the known protein and tRNA sequences. The 63.4-kilodalton monomeric enzyme consists of four domains arranged to give an elongated molecule with an axial ratio greater than 3 to 1. Its interactions with the tRNA extend from the anticodon to the acceptor stem along the entire inside of the L of the tRNA. The complexed tRNA retains the overall conformation of the yeast phenylalanine tRNA (tRNA(Phe] with two major differences: the 3' acceptor strand of tRNA(Gln) makes a hairpin turn toward the inside of the L, with the disruption of the final base pair of the acceptor stem, and the anticodon loop adopts a conformation not seen in any of the previously determined tRNA structures. Specific recognition elements identified so far include (i) enzyme contacts with the 2-amino groups of guanine via the tRNA minor groove in the acceptor stem at G2 and G3; (ii) interactions between the enzyme and the anticodon nucleotides; and (iii) the ability of the nucleotides G73 and U1.A72 of the cognate tRNA to assume a conformation stabilized by the protein at a lower free energy cost than noncognate sequences. The central domain of this synthetase binds ATP, glutamine, and the acceptor end of the tRNA as well as making specific interactions with the acceptor stem.2+t is

A cognate tRNA specific conformational change in glutaminyl‐tRNA synthetase and its implication for specificity

“…In neither case is there a significant change in the CD spectra. This is consistent with the conclusions drawn from X-ray crystallographic studies, where only significant distortion seen in the bound tRNA structure from the presumed free tRNA structure is in the acceptor end and anticodon loops, and modest distortions are seen elsewhere (Rould et a]., 1989). Figure 6 shows the far UV-CD spectra of the protein in a complex with ATP and tRNAG'" and with ATP alone.…”

A three‐dimensional structure model of the complex of glutamyl‐tRNA synthetase and its cognate tRNA

“…For example, in the X-ray structure of the GlnRS and tRNA GIn complex, the cytidine at position 74 (C74) of the tRNA is looped out and buried in a pocket of GlnRS, and the cytosine base forms a stack with Pro126 [9]. Therefore, it is possible that GluRS recognizes the C74 of tRNA °lu.…”

“…As for class I, Escherichia coli glutaminyl-tRNA synthetase (GlnRS) is the only one for which a crystal structure has been determined for the complex with the cognate tRNA [9,10]. Recently, we have solved the 3D structure of another class I synthetase, glutamyl-tRNA synthetase (GluRS) from Thermus thermophilus [11].…”

Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data

“…In our models, we assume that tRNAs maintain their crystallographically determined conformation when bound to the ribosome+ It is possible that tRNAs undergo significant conformational change when bound to the ribosome as suggested by the crystal structures of tRNA-synthetase complexes (Rould et al+, 1989;Ruff et al+, 1991) and by low-resolution cryoelectron microscopy difference maps of tRNA-ribosome complexes (Malhotra et al+, 1998)+ To understand the molecular mechanism of translation, it is essential to elucidate the mutual arrangement of tRNAs on the ribosome+ So far, at least a half-dozen different binding states of tRNA have been described (Green & Noller, 1997)+ In these studies, the tRNAs are bound in the A/A and P/P states, corresponding to the classical A and P sites+ They are in the S orientation with the P tRNA on the left and A tRNA on the right as viewed from the 30S toward the 50S subunit (Fig+ 7A)+ The anticodon stem-loops are at the bottom, interacting mainly with the 30S subunit and mRNA; the 39-CCA ends are at the top, interacting with the 50S subunit+ Although the 39 termini of the two tRNAs are insufficiently close to allow peptide bond formation, flexibility of their single-stranded 39-ACCA termini could allow a closer approach+ Placement of A-site tRNA on the right is consistent with the interaction of the EF-Tu ternary complex near the L7/L12 stalk at the right of the 50S subunit (Girshovich et al+, 1986;Stark et al+, 1997b)+ Also, in the crystal structure of the EF-Tu:GDPNP:tRNA ternary complex, the T-loop side of the A-site tRNA is in contact with EF-Tu (Nissen et al+, 1995)+ Therefore, this side cannot face P tRNA, consistent with the S orientation+ An important ribosomal function is translocation, the coordinated movement of the tRNA-mRNA complex within the ribosome following peptide bond formation (Kaziro, 1978;Spirin, 1985;Czworkowski & Moore, 1996;Wilson & Noller, 1998)+ During translocation, tRNAs move from right to left from A site to P site as shown in Figure 7A+ Our model predicts that translocation of A-site tRNA into the P site could be accomplished by a rotational movement of about 458 around an axis drawn from the 39-CCA end through the anticodon stemloop of the A-site tRNA, coupled with a translational movement of about 24+5 Å from right to left+ Interestingly, we do not detect any cleavages in the anticodon stem-loop region of P tRNA, consistent with the previous observation that the anticodon stem-loop of P tRNA is protected from hydroxyl radicals by the 30S subunit (Hüttenhofer & Noller, 1994), most likely by features of 16S rRNA that line the cleft of the 30S subunit+ Nor do we detect any cleavages in the 39-CCA end of P tRNA from Fe(II) tethered to the 59 terminus of A tRNA+ The 39 end of P tRNA may be shielded from hydroxyl radicals by its interactions with the 2250 loop (Samaha et al+, 1995) and other features of 23S rRNA+ These studies have focused on two particular sets of tRNA binding complexes+ There are currently believed to be as many as eight (or possibly more) identifiable binding states for tRNA …”