Measurement of Internal Movements within the 30 S Ribosomal Subunit Using Förster Resonance Energy Transfer

Hickerson, Robyn P.; Majumdar, Zigurts K.; Baucom, Albion; Clegg, Robert M.; Noller, Harry F.

doi:10.1016/j.jmb.2005.09.010

Cited by 62 publications

(83 citation statements)

References 46 publications

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“…A single cysteine mutant of S12 (L48C) was labeled with the donor (Cy3) dye and introduced into the 30S subunit using an in vitro reconstitution as previously described (34). A toeprinting translocation assay showed that, consistent with an earlier report (3), at least 50% of reconstituted ribosomes were able to form pretranslocation complexes and were active in translocation (Fig.…”

supporting

confidence: 73%

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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supporting

confidence: 73%

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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“…Tight-couple 70S ribosomes and ribosomal subunits were prepared from Escherichia coli MRE600 as previously described (Lancaster et al 2002;Hickerson et al 2005). Cy5-labeled protein S6 was incorporated into 30S subunits by in vitro reconstitution from purified 16S rRNA and the other 19 individually purified ribosomal proteins according to published procedures (Culver and Noller 1999;Hickerson et al 2005). Cy3-labeled protein L9 was incorporated into 50S subunits by partial reconstitution from 50S subunits carrying an L9 deletion, and doubly labeled 70S ribosomes were isolated using previously described procedures (Ermolenko et al 2007a).…”

Section: Methodsmentioning

confidence: 99%

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

Ermolenko

Cornish

et al. 2012

RNA

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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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“…Tight-couple 70S ribosomes and ribosomal subunits were prepared from Escherichia coli MRE600 as previously described (Lancaster et al 2002;Hickerson et al 2005). For toeprinting, wild-type 30S subunits were activated by incubation for 10 min at 42°C in buffer A (50 mM HepesÁKOH [pH 7.5], 20 mM MgCl 2 , 100 mM NH 4 Cl, 6 mM b-mercaptoethanol) and associated with a 1.5-molar excess of wild-type 50S subunits for 10 min at 37°C in buffer A.…”

Section: Methodsmentioning

confidence: 99%

Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome

Spiegel

Ermolenko²,

Noller³

2007

RNA

Self Cite

122

135

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Following peptide bond formation, transfer RNAs (tRNAs) and messenger RNA (mRNA) are translocated through the ribosome, a process catalyzed by elongation factor EF-G. Here, we have used a combination of chemical footprinting, peptidyl transferase activity assays, and mRNA toeprinting to monitor the effects of EF-G on the positions of tRNA and mRNA relative to the A, P, and E sites of the ribosome in the presence of GTP, GDP, GDPNP, and fusidic acid. Chemical footprinting experiments show that binding of EF-G in the presence of the non-hydrolyzable GTP analog GDPNP or GDPÁfusidic acid induces movement of a deacylated tRNA from the classical P/P state to the hybrid P/E state. Furthermore, stabilization of the hybrid P/E state by EF-G compromises P-site codon-anticodon interaction, causing frame-shifting. A deacylated tRNA bound to the P site and a peptidyltRNA in the A site are completely translocated to the E and P sites, respectively, in the presence of EF-G with GTP or GDPNP but not with EF-GÁGDP. Unexpectedly, translocation with EF-GÁGTP leads to dissociation of deacylated tRNA from the E site, while tRNA remains bound in the presence of EF-GÁGDPNP, suggesting that dissociation of tRNA from the E site is promoted by GTP hydrolysis and/or EF-G release. Our results show that binding of EF-G in the presence of GDPNP or GDPÁfusidic acid stabilizes the ribosomal intermediate hybrid state, but that complete translocation is supported only by EF-GÁGTP or EF-GÁGDPNP.

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Measurement of Internal Movements within the 30 S Ribosomal Subunit Using Förster Resonance Energy Transfer

Cited by 62 publications

References 46 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

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

Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome

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