Deletion of the SRL causes structural changes in 23S rRNA. Primer-extension analysis of 23S rRNA extracted from WT or ΔSRL 50S subunits probed with D, dimethylsufate, or K, kethoxal. A and G are sequencing lanes; (−) unmodified subunits. Arrows indicate bases with altered reactivity in ΔSRL 50S subunits as compared to WT.

ΔSRL 50S subunits are missing ribosomal protein L16. (A) SDS gel of proteins extracted from affinity-purified WT and ΔSRL 50S subunits; arrow indicates the missing protein band. (B) Two-dimensional protein gel of WT and ΔSRL 50S subunits; protein L16 is circled. (C) Denaturing gel of rRNA extracted from 50S subunits shows that 5S rRNA is present in normal amounts in ΔSRL subunits.

ΔSRL 50S subunits are inactive in protein synthesis, but can associate with 30S subunits. (A) Poly-U mRNA directed [¹⁴C]-Phe incorporation was measured for ribosomes containing WT (■) or ΔSRL (▼) 50S subunits. Sucrose density-gradient sedimentation of WT and ΔSRL 50S subunits in the absence (B) or presence (C) of 30S subunits. Arrows indicate the position of 30S subunits, 50S subunits, or 70S ribosomes.

ΔSRL 50S subunits bind P-site tRNA to WT levels, but are defective in the puromycin reaction. (A) 50S subunit stimulation of P-site tRNA binding to 30S subunits (black bars) and puromycin reactivity (gray bars) were assayed in complexes containing [³⁵S]-fMet-tRNA^fMet, mRNA, and 30S subunits formed in the presence or absence of WT or ΔSRL 50S subunits as indicated. (B) ΔSRL (▲) and WT (■) 50S subunits bound to [³⁵S]-fMet-tRNA^fMet were reacted with increasing concentrations of puromycin. The results were fit to a single exponential curve and are also shown as a double-reciprocal plot (inset).

Deletion of the SRL confers a dominant-lethal phenotype. (A) Wild-type (WT) sequence of 23S rRNA nucleotides 2650–2670 and that of the SRL deletion mutant (ΔSRL), in which nucleotides 2653–2667 (the SRL) were replaced with a GAAA tetraloop. (B) Primer-extension analysis of 23S rRNA from affinity-purified WT and ΔSRL 50S subunits probed with either D, dimethylsulfate, or K, kethoxal. Lanes A and G are sequencing lanes; (−) unmodified subunits. (C) E. coli strains that express either WT or ΔSRL MS2-tagged 50S subunits from plasmid, via a heat-inducible promoter, were grown at 30°C, spotted on plates, and incubated at 30°C or 42°C.

Disrupted interactions within and between the front and back layers of the ΔSRL-affected region. Protected (orange) and enhanced (blue and pink) nucleotides are shown as in Figure 8; 23S rRNA (gray) helix numbers are indicated. (A) The front layer as seen in the 50S subunit crown view (Fig. 7B). (C) The corresponding back layer, with helix 95 (yellow) and the SRL (red) indicated. B and D are 180° rotations of A and C. (E) Secondary structure of 23S rRNA showing the front (cyan) and back (pink) layers; lines indicate tertiary interactions within the front (blue) or back (magenta) layers or between layers (orange) that are disrupted in the ΔSRL 50S subunits.

Detailed stereo views of the ΔSRL-affected region of the 50S subunit. (A) The region of 23S rRNA indicated in dark blue in Figure 7B is shown (gray). Bases that are protected (orange) or enhanced (blue and pink) in ΔSRL 50S subunits are indicated as spheres placed at the N1 positions of A or G or the N3 of C; enhanced nucleotides that interact directly with protein L16 are shown in pink. P-tRNA (orange), A-tRNA (yellow), proteins L6 and L16 (pink), helix 95 (yellow), and the SRL (red) are indicated. (B) As in A, with tRNAs removed for clarity. (C) 23S rRNA helix numbers and domains are indicated: domain II (blue), V (purple and orange), and VI (yellow); and the SRL (red). D and E are rotated 180° around the vertical axis relative to B and C.

ΔSRL 50S subunits are defective in EF-Tu-dependent and nonenzymatic A-site tRNA binding and in interaction with EF-G. (A) [³H]-Phe-tRNA^Phe was bound to the A site of ribosomal complexes containing mRNA and fMet-tRNA^fMet under either EF-Tu-dependent (gray and hatched bars, 7 mM Mg), or nonenzymatic (black bars, 20 mM Mg) conditions. A-site binding was normalized to the highest level obtained by WT ribosomes, which corresponds to 0.95 [³H]-Phe-tRNA^Phe per ribosome (EF-Tu-dependent) and 0.8 per ribosome (nonenzymatic). (B) Toeprint analysis of tRNA binding and translocation using m301 mRNA; ribosomal complexes contain tRNA and factors as indicated. Reverse transcriptase stops (arrows) correspond to the indicated codons in the P site, and the doublet band indicates A-site-bound tRNA (*). (C) Ribosome-dependent EF-G·GTP hydrolysis. ΔSRL (triangle) or WT (square) vacant ribosomes (filled symbols) or those containing polyU mRNA and tRNA^Phe (open symbols) were incubated in the presence of EF-G and [³²P]-γ-GTP. (D) EF-G binding. Quenching of fluorescein-labeled EF-G was measured with increasing concentrations of ΔSRL (▲) or WT (■) ribosomal complexes containing mRNA and tRNA^fMet.

Nucleotides affected by the SRL deletion are widely distributed in the 23S rRNA secondary structure, but localized within and around the functional core of the 50S subunit. (A) Secondary structure of 23S rRNA indicating nucleotides in ΔSRL 50S subunits that show strongly enhanced (●), weakly enhanced (∙), or reduced (○) reactivity to structure probes; the SRL (h95) is indicated in red. (B) 23S rRNA regions highlighted in A (dark black line) were mapped onto the 50S subunit of the E. coli ribosome crystal structure (Schuwirth et al. 2005), and are shown in dark blue. Protein L16 (pink), 23S rRNA helix 95 (yellow), and the SRL (red) are indicated; other ribosomal proteins are light blue and 5S and 23S rRNA are gray. P-site (orange) and A-site (yellow) tRNAs were docked onto the structure using coordinates from Yusupov et al. (2001) and Korostelev et al. (2006).