Interaction of Thiostrepton and Elongation Factor-G with the Ribosomal Protein L11-binding Domain

William S Bowen,J Stephen Lodmell,Walter E Hill,Emanuel J Murgola,Natalya Van Dyke

doi:10.1074/jbc.m407008200

Abstract

Ribosomal protein L11 and the L11 binding region of ribosomal RNA constitute an important domain involved in active functions of the ribosome during translation. We studied the effects of L11 knock-out and truncation mutations on the structure of the rRNA in this region and on its interactions with a translation elongation factor and the antibiotic thiostrepton. The results indicated that the structure of the L11-binding rRNA becomes conformationally flexible when ribosomes lack the entire L11 protein, but not when the C-terminal domain is present on ribosomes. Probing wild type and mutant ribosomes in the presence of the antibiotic thiostrepton and elongation factor-G (EF-G) rigorously localized the binding cleft of thiostrepton and suggested a role for the rRNA in the L11-binding domain in modulating factor binding. Our results also provide evidence that the structure of the rRNA stabilized by the C-terminal domain of L11 is necessary to stabilize EF-G binding in the post-translocation state, and thiostrepton may modulate this structure in a manner that interferes with the ribosome-EF-G interaction. The implications for recent models of thiostrepton activity and factor interactions are discussed.

Highlights

The region of the prokaryotic 50S ribosomal subunit associated with interactions of ribosome-dependent GTPase proteins such as elongation factors-G and -Tu (EF-G1 and EF-Tu), initiation factor-2, as well as with interactions with release factors-1 and -2 (RF1 and RF2) during translation is referred to as the GTPase-associated center or region [1]
Our results provide evidence that the structure of the rRNA stabilized by the C-terminal domain of L11 is necessary to stabilize elongation factor-G (EF-G) binding in the post-translocation state, and thiostrepton may modulate this structure in a manner that interferes with the ribosome-EF-G interaction
Chemical Probing of L11 Mutant Ribosomes—To determine whether the loss or truncation of L11 induced the loss of other ribosomal proteins, the adjacent pentameric complex (L10-(L7/L12)2) [2, 25, 35] from ribosomes, we compared the reactivities of several nucleotides in the region associated with pentameric complex binding, including C1044, A1046, G1047, A1050, G1110, A1111, and G1112

Summary

EXPERIMENTAL PROCEDURES

L11 Mutant Strains—Bacterial strains used in the study were derived from E. coli K-12 (Table I). 0.5 ␮M 70S wild type or mutant ribosomes in 1ϫ H50M7K100D1 buffer were incubated for 10 min at 25 °C with 5 ␮g of poly-U mRNA (Sigma), followed by 10 min at 25 °C with 1.5 ␮M deacylated tRNAPhe from E. coli (Sigma). To these complexes were added either 0.5 mM GDP and 0.2 mM fusidic acid (Sigma) or 0.5 mM GDPNP (Sigma), followed by addition of EF-G to 2.5 ␮M and incubation at 25 °C for 10 min. Preparation of rRNA and primer extension analysis for probing reactions was as above for thiostrepton experiments

RESULTS

37 Ϯ 6 21 Ϯ 4 24 Ϯ 5 26 Ϯ 5 16 Ϯ 3 20 Ϯ 5 17 Ϯ 5

DISCUSSION