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Stm1p is a Saccharomyces cerevisiae protein that is primarily associated with cytosolic 80S ribosomes and polysomes. Several lines of evidence suggest that Stm1p plays a role in translation under nutrient stress conditions, although its mechanism of action is not yet known. In this study, we show that yeast lacking Stm1p (stm1Δ) are hypersensitive to the translation inhibitor anisomycin, which affects the peptidyl transferase reaction in translation elongation, but show little hypersensitivity to other translation inhibitors such as paromomycin and hygromycin B, which affect translation fidelity. Ribosomes isolated from stm1Δ yeast have intrinsically elevated levels of eukaryotic elongation factor 3 (eEF3) associated with them. Overexpression of eEF3 in cells lacking Stm1p results in a growth defect phenotype and increased anisomycin sensitivity. In addition, ribosomes with increased levels of Stm1p exhibit decreased association with eEF3. Taken together, our data indicate that Stm1p plays a complementary role to eEF3 in translation.

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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...