Catalysis of Ribosomal Translocation by Sparsomycin

Fredrick, Kurt; Noller, Harry F.

doi:10.1126/science.1084571

Cited by 134 publications

(154 citation statements)

References 32 publications

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“…The mRNA m291, ribosomes, EF-Tu, aminoacylated tRNAs, and reconstituted ribosomes were prepared as previously described (3,53,54). Single-molecule FRET measurements were taken using a prism-type total internal reflection (TIR) microscope as described (17).…”

Section: Methodsmentioning

confidence: 99%

Following movement of domain IV of elongation factor G during ribosomal translocation

Salsi

Farah

Dann

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Translocation of mRNA and tRNAs through the ribosome is catalyzed by a universally conserved elongation factor (EF-G in prokaryotes and EF-2 in eukaryotes). Previous studies have suggested that ribosome-bound EF-G undergoes significant structural rearrangements. Here, we follow the movement of domain IV of EF-G, which is critical for the catalysis of translocation, relative to protein S12 of the small ribosomal subunit using single-molecule FRET. We show that ribosome-bound EF-G adopts distinct conformations corresponding to the pre- and posttranslocation states of the ribosome. Our results suggest that, upon ribosomal translocation, domain IV of EF-G moves toward the A site of the small ribosomal subunit and facilitates the movement of peptidyl-tRNA from the A to the P site. We found no evidence of direct coupling between the observed movement of domain IV of EF-G and GTP hydrolysis. In addition, our results suggest that the pretranslocation conformation of the EF-G–ribosome complex is significantly less stable than the posttranslocation conformation. Hence, the structural rearrangement of EF-G makes a considerable energetic contribution to promoting tRNA translocation.

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Section: Methodsmentioning

confidence: 99%

Following movement of domain IV of elongation factor G during ribosomal translocation

Salsi

Farah

Dann

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Although this phenomenon has previously been referred to as inaccurate translocation 23 , it is more consistent with explanations invoking low complex stability and tRNA rebinding events that occur without the initial bias of ordered tRNA binding (discussed in ref. 17 ). Thus, in the sparsomycin-catalyzed reaction, peptidyl-tRNA binding is stabilized by sparsomycin 19 , whereas in the absence of sparsomycin, peptidyl-tRNA falls off the ribosome after EF-Gdependent translocation and rebinds in a preferred position on the mRNA.…”

Section: Effects On Sparsomycin-dependent Translocationmentioning

confidence: 99%

“…17 . 32 P-labeled primer (0.6 μM of 5′-TTTATCTTCAGAAGAAAAACC-3′) complementary to the 3′end of m301 mRNA was annealed to mRNA template (8 μM) at 65 °C, incubated for 3 min and snap-cooled on ice.…”

Section: Toeprint Assaymentioning

confidence: 99%

The hybrid state of tRNA binding is an authentic translation elongation intermediate

Dorner

Brunelle

Sharma

et al. 2006

Nat Struct Mol Biol

121

140

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The GTPase elongation factor (EF)-G is responsible for promoting the translocation of the messenger RNA-transfer RNA complex on the ribosome, thus opening up the A site for the next aminoacyltRNA. Chemical modification and cryo-EM studies have indicated that tRNAs can bind the ribosome in an alternative 'hybrid' state after peptidyl transfer and before translocation, though the relevance of this state during translation elongation has been a subject of debate. Here, using pre-steady-state kinetic approaches and mutant analysis, we show that translocation by EF-G is most efficient when tRNAs are bound in a hybrid state, supporting the argument that this state is an authentic intermediate during translation.Translation elongation is the multistep process performed by the ribosome to sequentially add mRNA-encoded amino acids to the growing polypeptide chain. There are three iterated steps performed by the ribosome during the elongation cycle: (i) a tRNA-selection step in which the ribosome and elongation factor (EF)-Tu select the next aminoacyl-tRNA to enter the cycle; (ii) peptide-bond formation catalyzed in the active site of the large ribosomal subunit; and (iii) translocation of the mRNA-tRNA complex through the subunit interface region, facilitated by EF-G, with associated GTP hydrolysis. The tRNA substrates are at the heart of each of these steps, where they have key functional roles 1-3 .Early studies on the ribosome identified binding sites for two tRNA substrates, the A site for the aminoacyl-tRNA and the P site for the peptidyl-tRNA. Further biochemistry eventually identified a third site, the E or exit site, where deacylated tRNAs bind after loss of the peptidyl moiety and before release into solution 4 . An understanding of the order and timing with which tRNA substrates occupy these three sites (on both the large and small subunits) during the elongation cycle is central to a detailed molecular view of the process of translation.The hybrid state of tRNA binding on the ribosome was first proposed in the 1960s as an elegant way to understand how controlled tRNA movement through the ribosome might be accomplished 5 . The basic feature of such a hybrid state of binding is that tRNAs would move independently with respect to the two subunits of the ribosome during the steps of the translation cycle. Fluorescence studies provided early biochemical clues that such a state of tRNA binding might be populated during translation 6 , and subsequent chemical-modification analysis provided clear and detailed experimental support for the hybrid state 7 . These studies Correspondence should be addressed to R.G. (ragreen@jhmi.edu).. Note: Supplementary information is available on the Nature Structural & Molecular Biology website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.Published online at http://www.nature.com/nsmb/ Reprints and permissions information is available online at http://www.nature.com/reprints/index.html NIH Public Access Fig. 1a). Thus, 'hybr...

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“…Previously, an intriguing translocation phenomenon was discovered: The antibiotic sparsomycin, a peptidyl transferase inhibitor that binds to the large ribosomal subunit, was found to induce efficient translocation of tRNA and mRNA on the small subunit (Fredrick and Noller 2003). Early studies on the inhibitory action of sparsomycin showed that it increases the affinity of tRNA for the P site while interfering with binding to the A site (Monro et al 1969;Pestka 1969;Lazaro et al 1991).…”

Section: Introductionmentioning

confidence: 99%

Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation

Ermolenko

Cornish

et al. 2012

RNA

Self Cite

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In the absence of elongation factor EF-G, ribosomes undergo spontaneous, thermally driven fluctuation between the pretranslocation (classical) and intermediate (hybrid) states of translocation. These fluctuations do not result in productive mRNA translocation. Extending previous findings that the antibiotic sparsomycin induces translocation, we identify additional peptidyl transferase inhibitors that trigger productive mRNA translocation. We find that antibiotics that bind the peptidyl transferase A site induce mRNA translocation, whereas those that do not occupy the A site fail to induce translocation. Using single-molecule FRET, we show that translocation-inducing antibiotics do not accelerate intersubunit rotation, but act solely by converting the intrinsic, thermally driven dynamics of the ribosome into translocation. Our results support the idea that the ribosome is a Brownian ratchet machine, whose intrinsic dynamics can be rectified into unidirectional translocation by ligand binding.

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Catalysis of Ribosomal Translocation by Sparsomycin

Cited by 134 publications

References 32 publications

Following movement of domain IV of elongation factor G during ribosomal translocation

Following movement of domain IV of elongation factor G during ribosomal translocation

The hybrid state of tRNA binding is an authentic translation elongation intermediate

Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation

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