Rapid Kinetic Analysis of EF-G-dependent mRNA Translocation in the Ribosome

Studer, Sean M.; Feinberg, Jason S.; Joseph, Simpson

doi:10.1016/s0022-2836(03)00146-3

Cited by 104 publications

(176 citation statements)

References 52 publications

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“…This value is consistent with previous reports (24,53). Interestingly, single-molecule time traces reveal both high-and low-FRET-state tRNA configurations after translocation.…”

Section: Surface-immobilized Ribosomes Are Highly Active In Peptide Bondsupporting

confidence: 93%

tRNA dynamics on the ribosome during translation

Blanchard

Kim

Gonzalez

et al. 2004

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

454

575

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Using single-molecule fluorescence spectroscopy, time-resolved conformational changes between fluorescently labeled tRNA have been characterized within surface-immobilized ribosomes proceeding through a complete cycle of translation elongation. Fluorescence resonance energy transfer was used to observe aminoacyl-tRNA (aa-tRNA) stably accommodating into the aminoacyl site (A site) of the ribosome via a multistep, elongation factor-Tu dependent process. Subsequently, tRNA molecules, bound at the peptidyl site and A site, fluctuate between two configurations assigned as classical and hybrid states. The lifetime of classical and hybrid states, measured for complexes carrying aa-tRNA and peptidyl-tRNA at the A site, shows that peptide bond formation decreases the lifetime of the classical-state tRNA configuration by Ϸ6-fold. These data suggest that the growing peptide chain plays a role in modulating fluctuations between hybrid and classical states. Single-molecule fluorescence resonance energy transfer was also used to observe aa-tRNA accommodation coupled with elongation factor G-mediated translocation. Dynamic rearrangements in tRNA configuration are also observed subsequent to the translocation reaction. This work underscores the importance of dynamics in ribosome function and demonstrates single-particle enzymology in a system of more than two components. P rotein synthesis, catalyzed by the ribosome, is rapid, processive, and highly regulated. In bacteria, two RNA-protein subunits consisting of a small 30S subunit and a large 50S subunit assemble around a mRNA template into a roughly spherical 70S particle that translates the nucleotide sequence into a polypeptide chain through repetitive, codon-dependent binding of aminoacylated tRNA.The landmark structures of the 30S (1, 2), 50S (3, 4), and 70S (5) ribosomal particles provide a molecular basis for understanding ribosome function. tRNA molecules bind to the ribosome in a solvent-accessible channel at the subunit interface. Three binding sites for tRNA, called the aminoacyl site (A site), peptidyl site (P site), and exit site (E site), have been identified on both the large and small subunit (Fig. 1). Each tRNA is separated from its neighbor at the elbow region (the site of Ϸ90°b ending) by 25-45 Å (5, 6). The anticodon stem loops of A-and P-site tRNA form Watson-Crick base pairs with adjacent mRNA codons on the 30S subunit (5, 7), whereas the 3Ј CCA terminal residues of A-and P-site tRNAs base-pair with conserved ribosomal RNA (rRNA) loops (8-10) within the 50S subunit peptidyltransferase center, the site of peptide bond formation (11). Additional interactions with rRNA and ribosomal proteins position tRNA molecules on the ribosome (5).The essential components and principal steps of translation have been delineated through genetic, biochemical, and kinetic methods (12). The process of polypeptide elongation has been most extensively characterized (13). Binding of aminoacyl-tRNA (aa-tRNA) to the A site occurs as a ''ternary complex'' with the GTPase elongation ...

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“…This value is consistent with previous reports (24,53). Interestingly, single-molecule time traces reveal both high-and low-FRET-state tRNA configurations after translocation.…”

Section: Surface-immobilized Ribosomes Are Highly Active In Peptide Bondsupporting

confidence: 93%

tRNA dynamics on the ribosome during translation

Blanchard

Kim

Gonzalez

et al. 2004

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

454

575

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“…We next attempted to test whether this conformation is sampled by EF-G during translocation by flowing in EF-G·GTP·Fus to pretranslocation ribosomes while imaging. However, real-time observation of translocation is hampered by the fact that the rate of translocation is determined to be 5-20 s −1 (16,21,38,39). Thus, the dwell time of EF-G in the pretranslocation state (τ = 1/k) is expected to be 50-200 ms, which is near or below the time resolution of our smFRET measurements (100 ms).…”

Section: Ef-g Adopts Different Conformations In Pre-and Posttranslocamentioning

confidence: 65%

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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“…E. coli tRNA fMet and tRNA Phe were purchased from Sigma. The mRNA, 5Ј-AAGGAGGUAAAAAUGUUUGCU(N 6 )-3Ј, was synthesized by Dharmacon and conjugated at its 3Ј end with pyrene as described (16). Mant-labeled nucleotides were purchased from Invitrogen.…”

Section: Methodsmentioning

confidence: 99%

“…Single-turnover ribosomal translocation was monitored by the change in fluorescence of mRNA labeled with pyrene at its 3Ј end (16). A pretranslocation complex (0.5 M) was formed in 5 ml of buffer 3, similar to the above complex except substituting pyrene-mRNA (0.4 M).…”

Section: Methodsmentioning

confidence: 99%

“…To measure accurately the kinetics of translocation under single-turnover conditions, we took advantage of a fluorescence assay that monitors the movement of pyrene attached to the 3Ј end of a short mRNA that just spans the ribosome (16). A pretranslocational complex (0.5 M) containing pyrene-labeled mRNA and the same two tRNAs was rapidly mixed with wild type EF-G (1.25 M) and saturating GTP.…”

Section: Figure 1 Locations Of Mutated Residues In the G Subdomain Omentioning

confidence: 99%

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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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Rapid Kinetic Analysis of EF-G-dependent mRNA Translocation in the Ribosome

Cited by 104 publications

References 52 publications

tRNA dynamics on the ribosome during translation

tRNA dynamics on the ribosome during translation

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

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

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