Locking and Unlocking of Ribosomal Motions

Valle, Mikel; Zavialov, Andrey V.; Sengupta, Jayati; Rawat, U.; Ehrenberg, Måns; Frank, Joachim

doi:10.1016/s0092-8674(03)00476-8

Cited by 572 publications

(685 citation statements)

References 45 publications

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“…Taken together, we propose that aminoglycoside binding to the ribosome makes it difficult for subunits to move relative to each other (8,38), resulting in inhibition of translocation (2,10,16,24). On the other hand, alternative possibilities of the mechanism of paromomycin inhibition on the translocation not related to the current observation are valid.…”

supporting

confidence: 48%

Inhibition of Antiassociation Activity of Translation Initiation Factor 3 by Paromomycin

Hirokawa

Kaji

2007

Antimicrob Agents Chemother

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The effect of paromomycin on the interaction of ribosomal subunits was studied. Paromomycin inhibited the antiassociation activity of initiation factor 3 (IF3). Furthermore, ribosomal subunits were associated to form 70S ribosomes by paromomycin even in the presence of 1 mM Mg 2؉ . Paromomycin did not inhibit the binding of IF3 to the 30S ribosomal subunits. On the other hand, IF3 bound to the 30S subunits was expelled by paromomycin-induced subunit association (70S formation). These results indicate that the stabilization of 70S ribosomes by paromomycin may in part be responsible for its inhibitory effects on translocation and ribosome recycling.Aminoglycosides cause miscoding (6, 18), stabilization of 70S ribosomes (4, 26), inhibition of translocation (2,10,16,24), alanylation of transfer mRNA (37), and recycling of ribosomes (12,13,17). Although the mechanisms of the miscoding effect of paromomycin have been worked out previously (27)(28)(29), the exact mechanism of the effect of paromomycin on the translocation and recycling steps remains obscure. We investigated the effect of paromomycin on the interaction of subunits which plays an important role in both of these steps. We show that paromomycin inhibits the antiassociation activity of initiation factor 3 (IF3), which is an important component of the disassembly reaction of posttermination ribosomal complexes (14). This inhibitory effect comes from the fact that paromomycin strengthens the interaction between ribosomal subunits and even induces association of the subunits at low Mg 2ϩ concentrations. Upon the association of subunits by paromomycin, the 30S-bound IF3 is displaced from the ribosomes. A possible mechanism of the inhibitory action of paromomycin on the translocation step is discussed. MATERIALS AND METHODSBuffers. Buffer J consisted of 10 mM Tris-HCl, pH 7.4, 10 mM MgSO 4 , 50 mM KCl, and 1 mM dithiothreitol (DTT). Buffer R consisted of 10 mM Tris-HCl, pH 7.4, 8.2 mM MgSO 4 , 84 mM NH 4 Cl, and 0.5 mM DTT. Buffer S consisted of 10 mM Tris-HCl, pH 7.4, 1 mM MgSO 4 , 84 mM NH 4 Cl, and 0.5 mM DTT. Buffer S2 consisted of 10 mM Tris-HCl, pH 7.4, 4 mM MgSO 4 , 84 mM NH 4 Cl, and 0.5 mM DTT. Buffer S3 consisted of 10 mM Tris-HCl, pH 7.4, 6 mM MgSO 4 , 84 mM NH 4 Cl, and 0.5 mM DTT.Ribosomes and factors. Vacant ribosomes were prepared from Escherichia coli MRE600 (purchased from the University of Alabama Fermentation Facility, Birmingham) as described previously (21). Ribosome recycling factor (RRF) and elongation factor G (EF-G) were purified as described previously (11, 21) from E. coli DH5␣ harboring plasmid pRR2 (34) and E. coli JM83 harboring plasmid pECEG (15) (obtained from P. March), respectively. IF3 was purified from E. coli XL1-Blue harboring a plasmid expressing His-IF3 (35) (obtained from T. Ueda).Sucrose density gradient ultracentrifugation (SDGC). Unless otherwise mentioned, 0.6 A 260 units (approximately 14 pmol) of 70S ribosomes (or the dissociated subunits) or 0.45 A 260 units (approximately 31 pmol) of isolated 30S subunits in 275 l...

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supporting

confidence: 48%

Inhibition of Antiassociation Activity of Translation Initiation Factor 3 by Paromomycin

Hirokawa

Kaji

2007

Antimicrob Agents Chemother

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“…Possibly, these interactions may allosterically alter the configurations of catalytic residues of the G domain, or may affect the positioning of another ribosomal component that directly contacts the GTP substrate. According to cryo-EM models, nucleotides of helix 95 (or Sarcin-Ricin loop) of 23 S RNA may directly contact the G domain (19,35).…”

Section: Discussionmentioning

confidence: 99%

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Nechifor

Murataliev

Wilson

2007

Journal of Biological Chemistry

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Protein L7/L12 of the bacterial ribosome plays an important role in activating the GTP hydrolytic activity of elongation factor G (EF-G), which promotes ribosomal translocation during protein synthesis. Previously, we cross-linked L7/L12 from two residues (209 and 231) flanking ␣-helix A G in the G subdomain of Escherichia coli EF-G. Here we report kinetic studies on the functional effects of mutating three neighboring glutamic acid residues (224, 228, and 231) to lysine, either singly or in combination. Two single mutations (E224K and E228K), both within helix A G , caused large defects in GTP hydrolysis and smaller defects in ribosomal translocation. Removal of L7/L12 from the ribosome strongly reduced the activities of wild type EF-G but had no effect on the activities of the E224K and E228K mutants. Together, these results provide evidence for functionally important interactions between helix A G of EF-G and L7/L12 of the ribosome.

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“…The proteins contain ATP/ADP-binding ABC domains, which convert chemical energy derived from binding of ATP or its hydrolysis into a 'powerstroke' of mechanical energy 6 . ABC proteins function as either homodimers or as twin-cassette proteins with two ABC domains within the same polypeptide.The ribosome exhibits very dynamic behaviour, such as the ratchet movement 7 or the movement of the L1 and the L7/L12 stalks [8][9][10][11] . Hence, an intriguing question is how the interaction of eEF3 with the ribosome is correlated with its dynamic properties as an ABC protein, and how the energy derived from binding/hydrolysis of ATP is used for its function.…”

mentioning

confidence: 99%

“…The ribosome exhibits very dynamic behaviour, such as the ratchet movement 7 or the movement of the L1 and the L7/L12 stalks [8][9][10][11] . Hence, an intriguing question is how the interaction of eEF3 with the ribosome is correlated with its dynamic properties as an ABC protein, and how the energy derived from binding/hydrolysis of ATP is used for its function.…”

mentioning

confidence: 99%

Structure of eEF3 and the mechanism of transfer RNA release from the E-site

Andersen

Becker

Blau

et al. 2006

Nature

152

166

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Elongation factor eEF3 is an ATPase that, in addition to the two canonical factors eEF1A and eEF2, serves an essential function in the translation cycle of fungi. eEF3 is required for the binding of the aminoacyl-tRNA-eEF1A-GTP ternary complex to the ribosomal A-site and has been suggested to facilitate the clearance of deacyl-tRNA from the E-site. Here we present the crystal structure of Saccharomyces cerevisiae eEF3, showing that it consists of an amino-terminal HEAT repeat domain, followed by a four-helix bundle and two ABC-type ATPase domains, with a chromodomain inserted in ABC2. Moreover, we present the cryo-electron microscopy structure of the ATP-bound form of eEF3 in complex with the post-translocational-state 80S ribosome from yeast. eEF3 uses an entirely new factor binding site near the ribosomal E-site, with the chromodomain likely to stabilize the ribosomal L1 stalk in an open conformation, thus allowing tRNA release.Protein synthesis requires, in general, only two canonical GTPase elongation factors. eEF1A (known as EF-Tu in prokaryotes) recruits cognate aminoacyl-tRNAs to the A-site of the ribosome, and, after peptidyl transfer, eEF2 (EF-G in prokaryotes) catalyses translocation of the messenger RNA and the transfer RNAs from the A-and P-sites to the P-and E-sites. In contrast to the canonical factors, eEF3 has an ATPase activity that is stimulated by ribosomes. It interacts with both ribosomal subunits 1-3 , competes with eEF2 for binding to ribosomes, and stimulates eEF1A-dependent binding of cognate aminoacyltRNA to the ribosomal A-site 1,4 . Because, according to the allosteric three-site model of the ribosomal elongation cycle, E-site release is required for efficient A-site binding, it has been suggested that eEF3 functions as a so-called 'E-site factor' 4,5 . Moreover, ATP hydrolysis by eEF3 is required in every elongation cycle to allow chasing of deacyltRNA from the E-site 4 .eEF3 belongs to the family of ABC (ATP-binding cassette) proteins that includes proteins involved in transport across membranes, DNA repair, and translation. The membrane proteins of this class especially represent important targets for development of novel therapeutic strategies. The proteins contain ATP/ADP-binding ABC domains, which convert chemical energy derived from binding of ATP or its hydrolysis into a 'powerstroke' of mechanical energy 6 . ABC proteins function as either homodimers or as twin-cassette proteins with two ABC domains within the same polypeptide.The ribosome exhibits very dynamic behaviour, such as the ratchet movement 7 or the movement of the L1 and the L7/L12 stalks [8][9][10][11] . Hence, an intriguing question is how the interaction of eEF3 with the ribosome is correlated with its dynamic properties as an ABC protein, and how the energy derived from binding/hydrolysis of ATP is used for its function. Crystal structure of eEF3Three crystal structures of residues 1-980 of eEF3 in the apo state (2.7 Å ), in complex with ADP (2.4 Å ), or in complex with the nonhydrolysable ATP analogue ADPNP (3...

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Locking and Unlocking of Ribosomal Motions

Cited by 572 publications

References 45 publications

Inhibition of Antiassociation Activity of Translation Initiation Factor 3 by Paromomycin

Inhibition of Antiassociation Activity of Translation Initiation Factor 3 by Paromomycin

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Structure of eEF3 and the mechanism of transfer RNA release from the E-site

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