Xabier Agirrezabala

Visualization of the Hybrid State of tRNA Binding Promoted by Spontaneous Ratcheting of the Ribosome

A crucial step in translation is the translocation of tRNAs through the ribosome. In the transition from one canonical site to the other, the tRNAs acquire intermediate configurations, so-called hybrid states. At this stage, the small subunit is rotated with respect to the large subunit, and the anticodon stem loops reside in the A and P sites of the small subunit, while the acceptor ends interact with the P and E sites of the large subunit. In this work, by means of cryo-EM and particle classification procedures, we visualize the hybrid state of both A/P and P/E tRNAs in an authentic factor-free ribosome complex during translocation. In addition, we show how the repositioning of the tRNAs goes hand in hand with the change in the interplay between S13, L1 stalk, L5, H68, H69, and H38 that is caused by the ratcheting of the small subunit.

Maturation of phage T7 involves structural modification of both shell and inner core components

The double‐stranded DNA bacteriophages are good model systems to understand basic biological processes such as the macromolecular interactions that take place during the virus assembly and maturation, or the behavior of molecular motors that function during the DNA packaging process. Using cryoelectron microscopy and single‐particle methodology, we have determined the structures of two phage T7 assemblies produced during its morphogenetic process, the DNA‐free prohead and the mature virion. The first structure reveals a complex assembly in the interior of the capsid, which involves the scaffolding, and the core complex, which plays an important role in DNA packaging and is located in one of the phage vertices. The reconstruction of the mature virion reveals important changes in the shell, now much larger and thinner, the disappearance of the scaffolding structure, and important rearrangements of the core complex, which now protrudes the shell and interacts with the tail. Some of these changes must originate by the pressure exerted by the DNA in the interior of the head.

Elongation in translation as a dynamic interaction among the ribosome, tRNA, and elongation factors EF-G and EF-Tu.

The ribosome is a complex macromolecular machine that translates the message encoded in the messenger RNA and synthesizes polypeptides by linking the individual amino acids carried by the cognate transfer RNAs (tRNAs). The protein elongation cycle, during which the tRNAs traverse the ribosome in a coordinated manner along a path of more than 100 A, is facilitated by large-scale rearrangements of the ribosome. These rearrangements go hand in hand with conformational changes of tRNA as well as elongation factors EF-Tu and EF-G - GTPases that catalyze tRNA delivery and translocation, respectively. This review focuses on the structural data related to the dynamics of the ribosomal machinery, which are the basis, in conjunction with existing biochemical, kinetic, and fluorescence resonance energy transfer data, of our knowledge of the decoding and translocation steps of protein elongation.

The cryo-EM structure of a complete 30S translation initiation complex from Escherichia coli.

Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNAfMet requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNAfMet. Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNAfMet, IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNAfMet, which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNAfMet induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation.

Structural characterization of mRNA-tRNA translocation intermediates.

Cryo-EM analysis of a wild-type Escherichia coli pretranslocational sample has revealed the presence of previously unseen intermediate substates of the bacterial ribosome during the first phase of translocation, characterized by intermediate intersubunit rotations, L1 stalk positions, and tRNA configurations. Furthermore, we describe the domain rearrangements in quantitative terms, which has allowed us to characterize the processivity and coordination of the conformational reorganization of the ribosome, along with the associated changes in tRNA ribosome-binding configuration. The results are consistent with the view of the ribosome as a molecular machine employing Brownian motion to reach a functionally productive state via a series of substates with incremental changes in conformation.

Structural insights into cognate versus near-cognate discrimination during decoding.

The structural basis of the tRNA selection process is investigated by cryo‐electron microscopy of ribosomes programmed with UGA codons and incubated with ternary complex (TC) containing the near‐cognate Trp‐tRNATrp in the presence of kirromycin. Going through more than 350 000 images and employing image classification procedures, we find ∼8% in which the TC is bound to the ribosome. The reconstructed 3D map provides a means to characterize the arrangement of the near‐cognate aa‐tRNA with respect to elongation factor Tu (EF‐Tu) and the ribosome, as well as the domain movements of the ribosome. One of the interesting findings is that near‐cognate tRNAs acceptor stem region is flexible and CCA end becomes disordered. The data bring direct structural insights into the induced‐fit mechanism of decoding by the ribosome, as the analysis of the interactions between small and large ribosomal subunit, aa‐tRNA and EF‐Tu and comparison with the cognate case (UGG codon) offers clues on how the conformational signals conveyed to the GTPase differ in the two cases.

Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM.

The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF‐Tu, which forms a ternary complex with aminoacyl(aa)‐tRNA. To study the binding modes of different aa‐tRNAs, we compared cryo‐EM maps of the kirromycin‐stalled ribosome bound with ternary complexes containing Phe‐tRNAPhe, Trp‐tRNATrp, or Leu‐tRNALeuI. The three maps suggest a common binding manner of cognate aa‐tRNAs in their specific binding with both the ribosome and EF‐Tu. All three aa‐tRNAs have the same ‘loaded spring’ conformation with a kink and twist between the D‐stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF‐Tu‐binding CCA end of aa‐tRNA, proposed to signal cognate codon–anticodon interaction to the GTPase centre and tune the accuracy of aa‐tRNA selection.

The ribosome triggers the stringent response by RelA via a highly distorted tRNA

The bacterial stringent response links nutrient starvation with the transcriptional control of genes. This process is initiated by the stringent factor RelA, which senses the presence of deacylated tRNA in the ribosome as a symptom of amino‐acid starvation to synthesize the alarmone (p)ppGpp. Here we report a cryo‐EM study of RelA bound to ribosomes bearing cognate, deacylated tRNA in the A‐site. The data show that RelA on the ribosome stabilizes an unusual distorted form of the tRNA, with the acceptor arm making contact with RelA and far from its normal location in the peptidyl transferase centre.

The near-atomic cryoEM structure of a flexible filamentous plant virus shows homology of its coat protein with nucleoproteins of animal viruses.

Flexible filamentous viruses include economically important plant pathogens. Their viral particles contain several hundred copies of a helically arrayed coat protein (CP) protecting a (+)ssRNA. We describe here a structure at 3.9 Å resolution, from electron cryomicroscopy, of Pepino mosaic virus (PepMV), a representative of the genus Potexvirus (family Alphaflexiviridae). Our results allow modeling of the CP and its interactions with viral RNA. The overall fold of PepMV CP resembles that of nucleoproteins (NPs) from the genus Phlebovirus (family Bunyaviridae), a group of enveloped (-)ssRNA viruses. The main difference between potexvirus CP and phlebovirus NP is in their C-terminal extensions, which appear to determine the characteristics of the distinct multimeric assemblies – a flexuous, helical rod or a loose ribonucleoprotein. The homology suggests gene transfer between eukaryotic (+) and (-)ssRNA viruses. DOI: http://dx.doi.org/10.7554/eLife.11795.001

Structural Insights into tRNA Dynamics on the Ribosome.

High-resolution structures at different stages, as well as biochemical, single molecule and computational approaches have highlighted the elasticity of tRNA molecules when bound to the ribosome. It is well acknowledged that the inherent structural flexibility of the tRNA lies at the heart of the protein synthesis process. Here, we review the recent advances and describe considerations that the conformational changes of the tRNA molecules offer about the mechanisms grounded in translation.