Dynamics of Recognition between tRNA and Elongation Factor Tu

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219

186

In translation, elongation factor Tu (EF-Tu) molecules deliver aminoacyl-tRNAs to the mRNA-programmed ribosome. The GTPase activity of EF-Tu is triggered by ribosome-induced conformational changes of the factor that play a pivotal role in the selection of the cognate aminoacyl-tRNAs. We present a 6.7-Å cryo-electron microscopy map of the aminoacyl-tRNA⅐EF-Tu⅐GDP⅐kirromycin-bound Escherichia coli ribosome, together with an atomic model of the complex obtained through molecular dynamics flexible fitting. The model reveals the conformational changes in the conserved GTPase switch regions of EF-Tu that trigger hydrolysis of GTP, along with key interactions, including those between the sarcin-ricin loop and the P loop of EF-Tu, and between the effector loop of EF-Tu and a conserved region of the 16S rRNA. Our data suggest that GTP hydrolysis on EF-Tu is controlled through a hydrophobic gate mechanism.decoding ͉ GTPase ͉ flexible fitting ͉ cryo-EM ͉ ternary complex

Section: Resultsmentioning

confidence: 53%

Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis

Villa

Sengupta

Trabuco

et al. 2009

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219

186

“…A small unresolved loop was modeled by using MODELLER (21). All simulations were performed in the NPT ensemble with CHARMM27 parameters (22) in NAMD2 (23) by using the protein/tRNA protocol in (24). The normalized covariance (correlation) and standard PCA of the MD simulations were performed by using CARMA (25).…”

Section: Methodsmentioning

confidence: 99%

Dynamical networks in tRNA:protein complexes

Sethi¹,

Eargle

Black³

et al. 2009

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738

1,091

Community network analysis derived from molecular dynamics simulations is used to identify and compare the signaling pathways in a bacterial glutamyl-tRNA synthetase (GluRS):tRNA Glu and an archaeal leucyl-tRNA synthetase (LeuRS):tRNA Leu complex. Although the 2 class I synthetases have remarkably different interactions with their cognate tRNAs, the allosteric networks for charging tRNA with the correct amino acid display considerable similarities. A dynamic contact map defines the edges connecting nodes (amino acids and nucleotides) in the physical network whose overall topology is presented as a network of communities, local substructures that are highly intraconnected, but loosely interconnected. Whereas nodes within a single community can communicate through many alternate pathways, the communication between monomers in different communities has to take place through a smaller number of critical edges or interactions. Consistent with this analysis, there are a large number of suboptimal paths that can be used for communication between the identity elements on the tRNAs and the catalytic site in the aaRS:tRNA complexes. Residues and nucleotides in the majority of pathways for intercommunity signal transmission are evolutionarily conserved and are predicted to be important for allosteric signaling. The same monomers are also found in a majority of the suboptimal paths. Modifying these residues or nucleotides has a large effect on the communication pathways in the protein:RNA complex consistent with kinetic data.aminoacyl-tRNA synthetase ͉ communication networks ͉ community ͉ suboptimal paths

“…To compute longrange electrostatic interactions, the Particle Mesh Ewald method (56) with a grid density of at least 1 Å −3 was used. The minimization and thermalization of all four complexes were performed as previously described (57). All systems were equilibrated for 10 ns followed by 40-ns production runs.…”

Section: Methodsmentioning

confidence: 99%

Structural insights into the effects of 2′-5′ linkages on the RNA duplex

Sheng

Engelhart

et al. 2014

The mixture of 2′-5′ and 3′-5′ linkages generated during the nonenzymatic replication of RNA has long been regarded as a central problem for the origin of the RNA world. However, we recently observed that both a ribozyme and an RNA aptamer retain considerable functionality in the presence of prebiotically plausible levels of linkage heterogeneity. To better understand the RNA structure and function in the presence of backbone linkage heterogeneity, we obtained high-resolution X-ray crystal structures of a native 10-mer RNA duplex (1.32 Å) and two variants: one containing one 2′-5′ linkage per strand (1.55 Å) and one containing three such linkages per strand (1.20 Å). We found that RNA duplexes adjust their local structures to accommodate the perturbation caused by 2′-5′ linkages, with the flanking nucleotides buffering the disruptive effects of the isomeric linkage and resulting in a minimally altered global structure. Although most 2′-linked sugars were in the expected 2′-endo conformation, some were partially or fully in the 3′-endo conformation, suggesting that the energy difference between these conformations was relatively small. Our structural and molecular dynamic studies also provide insight into the diminished thermal and chemical stability of the duplex state associated with the presence of 2′-5′ linkages. Our results contribute to the view that a low level of 2′-5′ substitution would not have been fatal in an early RNA world and may in contrast have been helpful for both the emergence of nonenzymatic RNA replication and the early evolution of functional RNAs.origin of life | backbone heterogeneity | X-ray crystallography T he capacity of RNA to act as both a carrier of genetic information and as a catalyst has led many to investigate its potential role as the first biopolymer (1-4). An early stage involving nonenzymatic replication simplifies RNA-first scenarios, but known nonenzymatic copying reactions generate a mixture of 3′-5′ and 2′-5′ backbone linkages because of the similar nucleophilicity and orientation of the 2′ and 3′ hydroxyl groups on ribose (Fig. 1). Although regioselectivity for the 3′-5′ linkage can be improved by using different metal ions or activated monomers, it reaches, at most, ∼90% (5-11). This lack of regiospecificity has been regarded as a central problem for the emergence of the RNA world, because the resulting backbone heterogeneity was expected to disrupt the folding, molecular recognition, and catalytic properties of functional RNAs. However, we recently observed that functional nucleic acid molecules can still evolve in the presence of nonheritable mixed DNA/RNA backbone heterogeneity (12), and known functional RNAs retain catalytic and ligand binding behavior in the presence of 2′-5′/3′-5′ backbone linkage heterogeneity (13).The well-known duplex-destabilizing property of 2′-5′ linkages can enable thermal strand separation of long RNA duplexes in the presence of the high Mg 2+ concentrations required for known prebiotic copying reactions (13-16). However, the mechanism r...