Binding Interaction between Tet(M) and the Ribosome: Requirements for Binding

ABSTRACT Tet(M) protein interacts with the protein biosynthesis machinery to render this process resistant to tetracycline by a mechanism which involves release of the antibiotic from the ribosome in a reaction dependent on GTP hydrolysis. To clarify this resistance mechanism further, the interaction of Tet(M) with the ribosome has been examined by using a gel filtration assay with radioactively labelled Tet(M) protein. The presence of GTP and 5′-guanylyl imido diphosphate, but not GDP, promoted Tet(M)-ribosome complex formation. Furthermore, thiostrepton, which inhibits the activities of elongation factor G (EF-G) and EF-Tu by binding to the ribosome, blocks stable Tet(M)-ribosome complex formation. Direct competition experiments show that Tet(M) and EF-G bind to overlapping sites on the ribosome.

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Elongation factor G initiates translocation through a power stroke

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1602668113 ◽

2016 ◽

Vol 113 (27) ◽

pp. 7515-7520 ◽

Cited By ~ 31

Author(s):

Chunlai Chen ◽

Xiaonan Cui ◽

John F. Beausang ◽

Haibo Zhang ◽

Ian Farrell ◽

...

Keyword(s):

Single Molecule ◽

Messenger Rna ◽

Elongation Factor ◽

Power Stroke ◽

Elongation Factor G ◽

Gtp Hydrolysis ◽

Guanosine Triphosphatase ◽

Prokaryotic Protein ◽

Domain Iii ◽

Factor G

During the translocation step of prokaryotic protein synthesis, elongation factor G (EF-G), a guanosine triphosphatase (GTPase), binds to the ribosomal PRE-translocation (PRE) complex and facilitates movement of transfer RNAs (tRNAs) and messenger RNA (mRNA) by one codon. Energy liberated by EF-G’s GTPase activity is necessary for EF-G to catalyze rapid and precise translocation. Whether this energy is used mainly to drive movements of the tRNAs and mRNA or to foster EF-G dissociation from the ribosome after translocation has been a long-lasting debate. Free EF-G, not bound to the ribosome, adopts quite different structures in its GTP and GDP forms. Structures of EF-G on the ribosome have been visualized at various intermediate steps along the translocation pathway, using antibiotics and nonhydolyzable GTP analogs to block translocation and to prolong the dwell time of EF-G on the ribosome. However, the structural dynamics of EF-G bound to the ribosome have not yet been described during normal, uninhibited translocation. Here, we report the rotational motions of EF-G domains during normal translocation detected by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy. Our study shows that EF-G has a small (∼10°) global rotational motion relative to the ribosome after GTP hydrolysis that exerts a force to unlock the ribosome. This is followed by a larger rotation within domain III of EF-G before its dissociation from the ribosome.

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Activation of GTP hydrolysis in mRNA-tRNA translocation by elongation factor G

Science Advances ◽

10.1126/sciadv.1500169 ◽

2015 ◽

Vol 1 (4) ◽

pp. e1500169 ◽

Cited By ~ 29

Author(s):

Wen Li ◽

Zheng Liu ◽

Ravi Kiran Koripella ◽

Robert Langlois ◽

Suparna Sanyal ◽

...

Keyword(s):

Elongation Factor ◽

Elongation Factor G ◽

Direct Role ◽

Gtp Hydrolysis ◽

Ribosomal Subunits ◽

Trna Translocation ◽

Gtpase Activation ◽

Guanosine Triphosphatase ◽

Factor G

During protein synthesis, elongation of the polypeptide chain by each amino acid is followed by a translocation step in which mRNA and transfer RNA (tRNA) are advanced by one codon. This crucial step is catalyzed by elongation factor G (EF-G), a guanosine triphosphatase (GTPase), and accompanied by a rotation between the two ribosomal subunits. A mutant of EF-G, H91A, renders the factor impaired in guanosine triphosphate (GTP) hydrolysis and thereby stabilizes it on the ribosome. We use cryogenic electron microscopy (cryo-EM) at near-atomic resolution to investigate two complexes formed by EF-G H91A in its GTP state with the ribosome, distinguished by the presence or absence of the intersubunit rotation. Comparison of these two structures argues in favor of a direct role of the conserved histidine in the switch II loop of EF-G in GTPase activation, and explains why GTP hydrolysis cannot proceed with EF-G bound to the unrotated form of the ribosome.

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Crystallographic studies of elongation factor G

Biochemistry and Cell Biology ◽

10.1139/o95-130 ◽

1995 ◽

Vol 73 (11-12) ◽

pp. 1209-1216 ◽

Cited By ~ 17

Author(s):

Anders Liljas ◽

Arnthor Ævarsson ◽

Salam Al-Karadaghi ◽

Maria Garber ◽

Julia Zheltonosova ◽

...

Keyword(s):

Crystal Structures ◽

Conformational Change ◽

Conformational Changes ◽

Elongation Factor ◽

Elongation Factors ◽

Elongation Factor G ◽

Conformational States ◽

Gtp Hydrolysis ◽

Large Conformational Change ◽

Factor G

The elongation factors G (EF-G) and Tu (EF-Tu) go through a number of conformation states in their functional cycles. Since they both are GTPases, have similar G domains and domains II, and have similar interactions with the nucleotides, then GTP hydrolysis must occur in similar ways. The crystal structures of two conformational states are known for EF-G and three are known for EF-Tu. The conformations of EF-G∙GDP and EF-Tu∙GTP are closely related. EF-Tu goes through a large conformational change upon GTP cleavage. This conformational change is to a large extent due to an altered interaction between the G domain and domains II and III. A number of kirromycin-resistant mutations are situated at the interface between domains I and III. The interface between the G domain and domain V in EF-G corresponds with this dynamic interface in EF-Tu. The contact area in EF-G is small and dominated by interactions between charged amino acids, which are part of a system that is observed to undergo conformational changes. Furthermore, a number of fusidic acid resistant mutants have been identified in this area. All of this evidence makes it likely that EF-G undergoes a large conformational change in its functional cycle. If the structures and conformational states of the elongation factors are related to a scheme in which the ribosome oscillates between two conformations, the pretranslocational and posttranslocational states, a model is arrived at in which EF-Tu drives the reaction in one direction and EF-G in the opposite. This may lead to the consequence that the GTP state of one factor is similar to the GDP state of the other. At the GTP hydrolysis state, the structures of the factors will be close to superimposable.Key words: elongation factor G, elongation factor Tu, crystal structures, conformational changes, ribosomal conformation.

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A Central Interdomain Protein Joint in Elongation Factor G Regulates Antibiotic Sensitivity, GTP Hydrolysis, and Ribosome Translocation

Journal of Biological Chemistry ◽

10.1074/jbc.m110.214056 ◽

2011 ◽

Vol 286 (24) ◽

pp. 21697-21705 ◽

Cited By ~ 8

Author(s):

Cristina Ticu ◽

Marat Murataliev ◽

Roxana Nechifor ◽

Kevin S. Wilson

Keyword(s):

Elongation Factor ◽

Antibiotic Sensitivity ◽

Elongation Factor G ◽

Gtp Hydrolysis ◽

Factor G

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Molecular Effects of Elongation Factor Ts and Trigger Factor on the Unfolding and Aggregation of Elongation Factor Tu Induced by the Prokaryotic Molecular Chaperone Hsp33

Biology ◽

10.3390/biology10111171 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1171

Author(s):

Minho Keum ◽

Dai Ito ◽

Mi-Seong Kim ◽

Yuxi Lin ◽

Kyeong-Hyeon Yoon ◽

...

Keyword(s):

Molecular Chaperones ◽

Protein Biosynthesis ◽

Gel Filtration ◽

Elongation Factor ◽

Substantial Effect ◽

Trigger Factor ◽

Titration Calorimetry ◽

Elongation Factor Tu ◽

Regulate Protein ◽

The Stability

Hsp33, a prokaryotic redox-regulated holding chaperone, has been recently identified to be able to exhibit an unfoldase and aggregase activity against elongation factor Tu (EF-Tu) in its reduced state. In this study, we investigated the effect of elongation factor Ts (EF-Ts) and trigger factor (TF) on Hsp33-mediated EF-Tu unfolding and aggregation using gel filtration, light scattering, circular dichroism, and isothermal titration calorimetry. We found that EF-Tu unfolding and subsequent aggregation induced by Hsp33 were evident even in its complex state with EF-Ts, which enhanced EF-Tu stability. In addition, although TF alone had no substantial effect on the stability of EF-Tu, it markedly amplified the Hsp33-mediated EF-Tu unfolding and aggregation. Collectively, the present results constitute the first example of synergistic unfoldase/aggregase activity of molecular chaperones and suggest that the stability of EF-Tu is modulated by a sophisticated network of molecular chaperones to regulate protein biosynthesis in cells under stress conditions.

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