The GTPase switch in ribosomal translocation

Journal of Biology BioMed Central Research news The GTPase switch in ribosomal translocation Pete Moore Published: 27 June 2005 Journal of Biology 2005, 4:7 The electronic version of this article is the complete one and can be found online at http://jbiol.com/content/4/2/7 © 2005 BioMed Central Ltd Information from careful measurements of the affinity of ribosome-associated proteins for GTP and GDP, and from structural analyses, suggests that elongation factor-G must assume three different structures during protein synthesis and that the ribosome itself acts as a guaninenucleotide exchange factor. Is it time to re-write the textbook description of translation? All subcellular units of macro-molecular machinery are complex, and an article in Journal of Biology [1] suggests that the way that ribosomes operate is more complex than had previously been suspected (see ‘The bottom line’ box for a summary of the work). Many factors assist the ribosome with the business of knitting amino acids together according to the instructions within mRNAs, so as to form proteins. Up until now it had been assumed that a critical factor, the GTPase elongation factor-G (EF-G), enters the ribosome in GTP-bound form, promoting translocation of peptidyl-tRNAs through the ribosome, and dissociating from the ribosome following GTP hydrolysis (see ‘Background’ box for further explanations and definitions). But working at Uppsala University in Sweden, a group of researchers now claim that EF-G in fact binds to the ribosome in GDP-bound form and that the arrival of GTP drives the ribosome into a recently described intermediate state. They also believe that a vital step in the way that energy is released from GTP molecules is stimulated by the ribosome, and not by some mysterious missing factor as was previously thought. Ribosomes are critical to every cell, and their structure and function are highly conserved in all organisms. They consist chiefly of two ribosomal subunits that clamp together like a clamshell, leaving a central channel The bottom line • The energy required for the translation of mRNA into peptide is provided by GTP, but it has been unclear exactly how and when the energy is released and put to use. • New purification and affinity studies show that the translation elongation factor EF-G has a 60-fold greater affinity for GDP than GTP, making it most probable that EF-G enters the ribosome in GDPbound form. • EF-G•GDP drives the ribosome into a recently described intermediary structural configuration during translocation. • The conditions within the ribosome cause GDP to be exchanged for GTP, with the ribosome itself appearing to act as a guanine-nucleotide exchange factor (GEF) for EF-G; EF-G then adopts a second conformation, driving translocation halfway. • GTP hydrolysis leads to a third structure of EF-G, which drives translocation to completion; EF-G adopts its entry structure and then leaves the ribosome. Journal of Biology 2005, 4:7 7.2 Journal of Biology 2005, Volume 4, Article 7 Moore http://jbiol.com/content/4/2/7 Amino acid Background E • The two ribosomal subunits differ between prokaryotes and eukaryotes, but both types include a smaller and a larger subunit, each contributing to the three sites - A, P and E - to which aminoacyltRNAs bind. P tRNA mRNA ACU AUG AUU UAA AUC P • GTP hydrolysis releases energy to drive the translocation of mRNA and tRNA through the ribosome, and is catalyzed by the GTPase elongation factor (EF-G). All known GTPases are acted on by guanine-nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP, and GTPase-activating proteins (GAPs) that stimulate the catalytic activity of the GTPase. The GTPase then cycles between GTP- and GDP-bound forms, acting as a molecular switch. • Molecular motor proteins, by contrast, utilize the hydrolysis of ATP or GTP to drive movement within cells, taking successive ‘steps’ with successive hydrolyses. • Translocation within the ribosome requires GTP hydrolysis, but it has not been clear precisely how the energy is used, or at what step the hydrolysis occurs. Numerous translation elongation factors, such as EF-G, cooperate with the ribosome to drive the process of growing a peptide (elongation). through which mRNA is drawn. Alongside the channel are three distinct sites formed by the two subunits (see Figure 1). First is the aminoacyl (A) site, where free-roaming charged tRNA molecules, bearing their respective amino acids, bind if their anticodon matches the sequence on the mRNA. Further into the channel is the peptidyl (P) site, which is occupied by a tRNA that had arrived moments earlier and now holds not only its own amino acid but through it the nascent peptide. This growing peptide now attaches to the amino acid held by the newly arrived tRNA in the A site, and the tRNAs are drawn through the channel. The tRNA that had previously been in the P site is now in the exit (E) site, the third in the series, and when the process cranks again the tRNA will be ejected into the cytosol. This much is uncontroversial. The debate begins when you look in more detail. The translocation of tRNA and mRNA through the ribosome requires energy, and this is supplied by the hydrolysis of GTP to GDP. Most textbooks show GTP being carried into the ribosome attached to EF-G. Now, however, Andrey Zavialov et al. [1] have shown that in the cytosol EF-G has a 60-fold greater affinity for GDP than GTP, so it is much more likely that EF-G carries GDP into the ribosome. Their study relied on complete purification of GDP away from GTP, whereas previous measurements have used GTP that contains some GDP contaminant (and vice versa). “It is almost certainly true that the preference of EF-G for GDP over GTP is much greater than the literature indicates, and hence that the concentration of EF-G•GDP in the cell is much higher relative to the concentration of EF-G•GTP than was previously thought,” says Professor Peter Moore Journal of Biology 2005, 4:7 A A Figure 1 The two-subunit ribosome with its three binding sites for tRNAs. See text for further details. of Yale University, who has a particular interest in the structure and function of RNAs and ribonucleoproteins. Building on this finding, Zavialov et al. [1] make another claim, although admitting that on this one they do not have 100% proof. “We believe that when EF-G•GDP enters the ribosome, this alone causes the ribosome to move into the hybrid configuration identified initially by Harry Noller,” says senior author Måns Ehrenberg, who is Professor of Molecular Biology at Uppsala University (see the ‘Behind the scenes’ box for more of the rationale for the work). Noller, who works at the University of California, Santa Cruz, showed in 1989 that during translocation the tRNAs are bound in hybrid sites linking the A site on the small subunit with the P site on the large, and the P site on the small subunit with the E site on the large [2]. Zavialov (...truncated)