The Conformational Properties of Elongation Factor G and the Mechanism of Translocation

Czworkowski, John; Moore, Peter B.

doi:10.1021/bi970610k

Cited by 61 publications

(50 citation statements)

References 48 publications

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“…for RNA binding (35)(36)(37)(38). This correlation between the different proteins provides indirect support for a role of the RNR motif of RNase P protein in RNA binding (17).…”

Section: Discussionmentioning

confidence: 83%

High-resolution structure of RNase P protein from Thermotoga maritima

Kazantsev

Krivenko

Harrington

et al. 2003

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

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The structure of RNase P protein from the hyperthermophilic bacterium Thermotoga maritima was determined at 1.2-Å resolution by using x-ray crystallography. This protein structure is from an ancestral-type RNase P and bears remarkable similarity to the recently determined structures of RNase P proteins from bacteria that have the distinct, Bacillus type of RNase P. These two types of protein span the extent of bacterial RNase P diversity, so the results generalize the structure of the bacterial RNase P protein. The broad phylogenetic conservation of structure and distribution of potential RNA-binding elements in the RNase P proteins indicate that all of these homologous proteins bind to their cognate RNAs primarily by interaction with the phylogenetically conserved core of the RNA. The protein is found to dimerize through an extensive, well-ordered interface. This dimerization may reflect a mechanism of thermal stability of the protein before assembly with the RNA moiety of the holoenzyme. RNase P catalyzes the divalent metal-dependent hydrolysis of a specific phosphodiester bond in pre-tRNAs, to release the 5Ј precursor sequences and produce the mature 5Ј ends of the tRNAs (see refs. 1-4 for reviews). All RNase P species studied to date are complex holoenzymes composed of one RNA and at least one protein component. The bacterial RNase P typically consists of a large RNA (350-400 nt; 100-130 kDa) and one small (Ϸ120 aa; 12-13 kDa) protein subunit. The active site of the bacterial RNase P is contained within the RNA. This is shown by the catalytic activity of the RNA in the absence of the protein moiety in vitro, at high ionic strength (5). At physiological ionic strength and in vivo, however, the protein component is required for pre-tRNA processing (5, 6).The role of the bacterial RNase P protein in the RNase P reaction is not entirely clear. The fact that it confers high activity on the RNA at physiological ionic strength suggests that the protein stabilizes the holoenzyme against electrostatic distortion (7). In vitro, the protein component of bacterial RNase P has been implicated in increasing the turnover rate of the enzyme (7), possibly by contribution to specific binding of the substrate over the product (8). Some mutational, enzymatic, and photoaffinity crosslinking studies have been interpreted to indicate that in the bacterial holoenzyme RNase P protein may form direct contacts with the 5Ј precursor sequence of the substrate pretRNA (9-11). The structural basis of any such interaction is not known. Moreover, there is no consistent structural model for the interaction between the protein and the RNase P RNA, despite several crosslink and footprint studies (11)(12)(13)(14).Phylogenetic comparative analysis has identified two major structural types of bacterial RNase P RNA (15). Both types have a homologous core structure that consists of about half the sequence lengths of the RNAs, but about half the sequence of each type of RNA has no homologous counterpart in the other RNA. The ancestral type (A type) ...

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“…for RNA binding (35)(36)(37)(38). This correlation between the different proteins provides indirect support for a role of the RNR motif of RNase P protein in RNA binding (17).…”

Section: Discussionmentioning

confidence: 83%

High-resolution structure of RNase P protein from Thermotoga maritima

Kazantsev

Krivenko

Harrington

et al. 2003

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

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“…How does EF-G catalyze the second step of translocation? It has been proposed that domain IV of EF-G occupies the 30S A site, displacing the anticodon stemloop of tRNA as the 30S subunit rotates clockwise from the hybrid state back to the classical state (Abel and Jurnak 1996;Czworkowski and Moore 1997). Alternatively, EF-GÁGTP (or EF-GÁGDPNP) could induce the formation of a second, yet undescribed, intermediate state of translocation, , and m291 mRNA to the ribosome.…”

Section: Discussionmentioning

confidence: 99%

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

Spiegel

Ermolenko²,

Noller³

2007

RNA

123

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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“…Because the sliding of the ribosome along the mRNA template is a mechanical output, analogous single-molecule measurements should provide information relevant toward the understanding of the molecular mechanism of protein synthesis. For instance, applying an external force to the mRNA and measuring the relationship between the rate of translocation and the force applied could help to distinguish between proposed mechanisms of translocation (Czworkowski and Moore 1997;Keller and Bustamante 2000;Wintermeyer and Rodnina 2000).…”

Section: Introductionmentioning

confidence: 99%

Protein synthesis by single ribosomes

Vanzi¹,

Vladimirov²,

Knudsen³

et al. 2003

RNA

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The ribosome is universally responsible for synthesizing proteins by translating the genetic code transcribed in mRNA into an amino acid sequence. Ribosomes use cellular accessory proteins, soluble transfer RNAs, and metabolic energy to accomplish the initiation, elongation, and termination of peptide synthesis. In translocating processively along the mRNA template during the elongation cycle, ribosomes act as supramolecular motors. Here we demonstrate that ribosomes adsorbed on a surface, as for mechanical or spectroscopic studies, are capable of polypeptide synthesis and that tethered particle analysis of fluorescent beads connected to ribosomes via polyuridylic acid can be used to estimate the rate of polyphenylalanine synthesis by individual ribosomes. This work opens the way for application of biophysical techniques, originally developed for the classical motor proteins, to the understanding of protein biosynthesis.

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The Conformational Properties of Elongation Factor G and the Mechanism of Translocation

Cited by 61 publications

References 48 publications

High-resolution structure of RNase P protein from Thermotoga maritima

High-resolution structure of RNase P protein from Thermotoga maritima

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

Protein synthesis by single ribosomes

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