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

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.The region of the prokaryotic 50S ribosomal subunit associated with interactions of ribosome-dependent GTPase proteins such as elongation factors-G and -Tu (EF-G 1 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). It contains three structural domains that are proximal on the 50S subunit: A pentameric complex (L10-(L7/L12) 2 ) that forms a protein stalk on the right shoulder of the 50S subunit (2, 3); the highly conserved sarcin-ricin stem-loop domain (nucleotides 2646 -2674 in Escherichia coli) (4); and ribosomal protein L11 and its binding site on 23S rRNA (L11-binding domain, nucleotides 1051-1102 in E. coli), adjacent to the binding site of the pentameric complex.Ribosomal protein L11 and its binding domain on 23S rRNA (L11-rRNA complex) are involved in thiazole peptide antibiotic binding (thiostrepton and micrococcin) (5, 6), have been implicated in binding of EF-G to the ribosome (7-9), and affect translation termination (10, 11). Ribosomes lacking L11 are resistant to thiostrepton, show severely reduced levels of protein synthesis activity in vitro, and bind thiostrepton poorly relative to wild type ribosomes (12, 13). Thiostrepton has been found to inhibit most factor-dependent processes of GTPase proteins (e.g. see Refs. 14 and 15; reviewed in Ref. 16) and the functions of some non-GTPase factors (RF1 (17), RF2 (17), and stringent factor, RelA (18)) on the prokaryotic ribosome. Kinetic studies by Rodnina et al. (14) indicate that thiostrepton binding to the L11-rRNA complex does not appear to interfere with factor binding or coupled GTPase activity on the ribosome, but it inhibits EF-G turnover subsequent to GTP hydrolysis (14). However, recent biochemical analysis by Camero...

Interactions of the N-terminal Domain of Ribosomal Protein L11 with Thiostrepton and rRNA

“…In particular, cryoelectron microscopy studies of EF-G⅐ribosome complexes show L11-NTD close to the A1067 hairpin loop before GTP hydrolysis, and displaced from this position by insertion of EF-G domain V between the L11-NTD and the RNA surface after GTP hydrolysis (11). The EF-G⅐GDP complex in these studies was stabilized on the ribosome by fusidic acid; similar complexes are known to protect the A1067 hairpin loop from reaction with chemical reagents (10,50,51). Insertion of thiostrepton between the L11-NTD and rRNA could thus sterically block EF-G interactions with the L11 BD region.…”

“…Unlabeled RNA of the same sequence (U1061A RNA) was transcribed from a plasmid DNA template by phage T7 RNA polymerase and purified by gel electrophoresis as described (29). Before use, RNAs were heated in renaturation buffer (10 (30). For binding studies with L11 and thiostrepton, a fixed concentration of thiostrepton,, usually 7 nM, was present during the titration.…”

“…A C-terminal domain of the protein, L11-C76, sits in the distorted minor groove of the L11 BD RNA (Fig. 1), binds the folded RNA with high affinity (8,9), and in Escherichia coli ribosomes, is essential for the RNA to form its native tertiary structure (10). Biochemical evidence and direct observation by cryoelectron microscopy have suggested that the N-terminal domain of L11 (L11-NTD) occupies different positions during the ribosome cycle and variously contacts elongation factor G (EF-G) (11), release factors 1 and 2 (12)(13)(14), and stringent factor (15).…”

“…Structure probing and NMR studies place thiostrepton near the hairpin loop containing A1067 (Fig. 1), but structural details of the thiostrepton-L11-rRNA complex are unresolved (10,18,19). Whether thiostrepton inhibits translation by competing with EF-G for ribosome binding or by stabilizing an EF-G⅐ribosome complex is also unresolved; there is experimental support for each model (20 -22).…”

Inactivation of Ribosomal Protein Genes in Bacillus subtilis Reveals Importance of Each Ribosomal Protein for Cell Proliferation and Cell Differentiation

“…L11 is one of the ribosomal proteins that have been well investigated, because mutations in the rplK gene often result in thiostrepton resistance (9,39,59). The N-terminal domain of L11, which interacts with the elongation factor G and the antibiotic thiostrepton, is implicated in the termination of translation (8,56). L11 is also required for the stringent response through the regulation of RelA activity, which synthesizes the signaling molecules GDP 3=-diphosphate (ppGpp) and GTP 3=-diphosphate (pppGpp), generally referred to as (p)ppGpp (11,59,61).…”