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

Abstract

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

Visualizing the protein synthesis machinery: New focus on the translational GTPase elongation factor Tu

“…The present study (2), together with the previous cryo-EM reconstructions of the same complex and the X-ray structures of the ribosome (4), identified several potentially important contacts that may help to induce and stabilize the active conformation of EF-Tu on the ribosome. On codon recognition, the 30S subunit switches to a closed conformation, in which the codon-anticodon duplex is stabilized, presumably involving an interaction with protein S12 (2,11). It is possible that the contact between the switch I region of EF-Tu and helices 8 and 14 in the 30S subunit requires the closed conformation of the 30S subunit, which would imply a direct link between changes in the decoding center and GTPase activation.…”

“…The reason for this difference is not clear, but it underlines the importance of high resolution in structural studies. Arg-58, which is located in the switch I region of EF-Tu in a position homologous to that of the catalytic arginine in G␣ proteins, is not essential for GTP hydrolysis by EF-Tu (12), in keeping with its position in the present structure (2).…”

“…However, in the ground state of EF-Tu, His-84 is oriented away from the water molecule; a simple rotation could place His-84 close to the buried water molecule, but a narrow hydrophobic gate formed by the side chains of Val-20 and Ile-61 would have to open to allow the movement (8). Villa et al (2) show that this is in fact the case: in their structure the gate is open, Ile-60 and the remainder of switch I are disordered, and His-84 is repositioned toward the nucleotide. The neighboring residue Gly-83 is important for the rearrangement of the switch II region during GTPase activation because of the conformational flexibility inherent to Gly residues, as well as in GTP hydrolysis itself, probably by helping to position the catalytic water by hydrogen bonding with the main chain oxygen of Gly-83 (10).…”

The Selenocysteine-specific Elongation Factor Contains a Novel and Multi-functional Domain

“…eEFSec GTPase Activity Is Regulated by Domain IV-The activation of translation GTPases, such as eEF1A, is achieved through conformational changes induced by the ribosome (30,31). Previous work with SelB, the Sec-specific elongation factor in bacteria, demonstrated that 70 S ribosomes increased the GTPase activity only when a bacterial SECIS element was present (32).…”

Partially Assembled Nucleosome Structures at Atomic Detail

“…In that sense, our approach bears an analogy with the use of low-resolution Cryo-EM in combination with atomistic modeling to produce fully atomistic models of large structures (93), including chromatin components (94,95). Unlike Cryo-EM, our approach can be used at ambient conditions.…”