Mapping the central fold of tRNA2fMet in the P site of the Escherichia coli ribosome

Abstract

4-Thiouridine (s4U), a photoreactive analog of uridine, was randomly incorporated into tRNA2(fMet) precursor molecules by transcription with T7 RNA polymerase. The s4U-containing transcripts were trimmed at their 5'-ends with RNase P RNA to yield mature tRNA2(fMet). The photoreactive tRNA2(fMet) derivatives were aminoacylated and bound to the P site of 70S ribosomes from Escherichia coli in the presence of a poly(A,G,U) template. Irradiation of the complexes at 300 nm resulted in the covalent cross-linking of tRNA2(fMet) to ribosomal proteins and rRNAs within both the 50S and 30S subunits. The labeled proteins were identified as L1, L27, and S19. 50S-subunit proteins L1 and L27 were attached to nucleotide U17 or U17.1 within the D loop of tRNA2(fMet), whereas 30S-subunit protein S19 was cross-linked to nucleotide U47 in the variable loop. Both of these sites occur in or near the central fold of the tRNA. These results permit us to map the D loop of P site-bound tRNA to the region between the central protuberance and the L1 ridge on the 50S ribosomal subunit, while the variable loop can be placed above the cleft on the head of the 30S subunit.

Direct localization of the tRNAs within the elongating ribosome by means of neutron scattering (proton-spin contrast-variation)

“…In both states, the mass centre of the ligand complex is located in the centre of the interface cavity separating both ribosomal subunits (Figure 6A and B) between the neck of the 30 S subunit ( Figure 6C) and the bottom of the central protuberance of the 50 S subunit ( Figure 6D). The central cavity has been proposed to accommodate the RNA ligands during protein biosynthesis (Frank et al, 1995 Rosen et al, 1993). The calculated position of the ligand mass centre based on neutron-scattering analysis supports current models, which are mainly based on the results of crosslinking studies (e.g.…”

The Structure of Ribosomal RNA: A Three‐Dimensional Jigsaw Puzzle

“…Fig. 9, below) described by Rosen et al [89] and Wower et al [90] can be satisfied by both tRNA configurations, and not just by the S configuration as these authors suggest [89]. On the other hand, the orientation of the A-site tRNA in Fig.…”

Ribosomal Protein L9 Interactions with 23 S rRNA: The Use of a Translational Bypass Assay to Study the Effect of Amino Acid Substitutions

“…Helix 80 is adjacent to helix 75 where the N domain of L9 binds ( Figure 6). Close proximity of the dihydrouridine loop (tRNA elbow) of P-site tRNA to L1 has been demonstrated with long-range crosslinking reagents of 10 and 19 Å at nt 20 (Podkowinski & Gornicki, 1991) and a shorter range reagent (4-thiouridine, 2 to 4 Å ) at nt 17 of the tRNA (Rosen et al, 1993). In addition, nt 8 of P-site tRNA (on the inside of the elbow) has been crosslinked to nt 2111 of the 23 S rRNA, using 4-thiouridine (Rinke-Appel et al, 1995).…”