Visualization of elongation factor Tu on the Escherichia coli ribosome

Abstract

The delivery of a specific amino acid to the translating ribosome is fundamental to protein synthesis. The binding of aminoacyl-transfer RNA to the ribosome is catalysed by the elongation factor Tu (EF-Tu). The elongation factor, the aminoacyl-tRNA and GTP form a stable 'ternary' complex that binds to the ribosome. We have used electron cryomicroscopy and angular reconstitution to visualize directly the kirromycin-stalled ternary complex in the A site of the 70S ribosome of Escherichia coli. Electron cryomicroscopy had previously given detailed ribosomal structures at 25 and 23 A resolution, and was used to determine the position of tRNAs on the ribosome. In particular, the structures of pre-translocational (tRNAs in A and P sites) and post-translocational ribosomes (P and E sites occupied) were both visualized at a resolution of approximately 20 A. Our three-dimensional reconstruction at 18 A resolution shows the ternary complex spanning the inter-subunit space with the acceptor domain of the tRNA reaching into the decoding centre. Domain 1 (the G domain) of the EF-Tu is bound both to the L7/L12 stalk and to the 50S body underneath the stalk, whereas domain 2 is oriented towards the S12 region on the 30S subunit.

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 …”

The cross-link from the upstream region of mRNA to ribosomal protein S7 is located in the C-terminal peptide: Experimental verification of a prediction from modeling studies

“…The recent rapid advances that have been made both in cryo-electron microscopy (cryo-EM) and X-ray crystallography of bacterial ribosomes or their subunits (e+g+, Stark et al+, 1997aStark et al+, , 1997bBan et al+, 1998;Malhotra et al+, 1998) have led to a correspondingly rapid advance in our understanding of the three-dimensional (3D) arrangement in situ of the ribosomal RNA and protein molecules+ In 1997 we published a model for the 16S rRNA , which was fitted to a cryo-EM reconstruction at 20 Å resolution of the Escherichia coli 70S ribosome carrying tRNAs at the ribosomal A and P sites (Stark et al+, 1997a)+ Subsequently, on the basis of the available RNAprotein interaction data (Mueller & Brimacombe, 1997b), we were able to fit the structure of ribosomal protein S7 as determined by X-ray crystallography (Hosaka et al+, 1997;Wimberly et al+, 1997) into our model in such a way as to satisfy both the biochemical data and the electron density of the EM reconstruction (Tanaka et al+, 1998)+ More recently, the 16S model has been refined to fit an EM reconstruction at 13 Å resolution (Brimacombe et al+, 2000), the latter being itself a refinement of the published EM reconstruction at 18 Å (Stark et al+, 1997b) of 70S ribosomes carrying an EF-Tu/tRNA ternary complex stalled with the antibiotic kirromycin+ In the refined 16S model, only minor changes needed to be made in the arrangement of the rRNA region interacting with S7 and in the positioning of the protein itself+ It is known that protein S7 can be cross-linked from sites in the upstream region of mRNA, close to the P site codon (Stade et al+, 1989;Dontsova et al+, 1991), as well as from sites in the anticodon loop of P sitebound tRNA (Wower et al+, 1993;Döring et al+, 1994)+ Our fitted structure (Tanaka et al+, 1998) made the strong prediction that the region of S7 involved in these crosslinks must be either in the b-sheet area of the protein (covering amino acids ;75-90) or at the extreme C-terminus (from amino acid ;145 within the C-terminal a-helix to the C-terminus itself at position 155)+ Here we demonstrate that the predominant cross-link from the upstream region of mRNA is indeed to the C-terminal region of the protein+ As in our previous studies (Dontsova et al+, 1991), an mRNA analogue related to the cro-mRNA from l-phage was prepared by T7 transcription from a suitable DNA template+ The mRNA sequence was GGGAAGGAGG UUGUAUGGACACCAAC{A 6 G{A 6 G{A 7 , thus containing a strong Shine-Dalgarno sequence close to the 59 end, the cro-mRNA UUGU spacer sequence, an AUG initiator codon, and an A-rich 39 sequence; the latter was included to enable the mRNA-protein cross-linked complex to be isolated by binding to oligo(dT)-cellulose (see below)+ The T7 transcription was carried out using 4-thio-UTP in place of "normal" UTP, except that a small ...…”

Macromolecular Mimicry in Translation Initiation: A Model for the Initiation Factor IF2 on the Ribosome

“…EF-Tu ! GTP as well as EF-G have been visualized on the surface of the E. coli 70S ribosome using cryo-electron microscopy (14,15), supporting the macromolecular mimicry hypothesis stated for elongation factors.…”

“…44). Moreover, using cryoelectronmicroscopy, new features of ribosomal particles and their complexes with EF-Tu and EF-G have been revealed (14,15,45). We have studied the IF2 domains responsible for recognition of the ribosome by means of assays in vitro using truncated forms of the factor as well as monoclonal antibodies (1,37).…”