Internal ribosome entry sites (IRESs) facilitate an alternative, end-independent pathway of translation initiation. A particular family of dicistroviral IRESs can assemble elongation-competent 80S ribosomal complexes in the absence of canonical initiation factors and initiator transfer RNA. We present here a cryo-EM reconstruction of a dicistroviral IRES bound to the 80S ribosome. The resolution of the cryo-EM reconstruction, in the subnanometer range, allowed the molecular structure of the complete IRES in its active, ribosome-bound state to be solved. The structure, harboring three pseudoknot-containing domains, each with a specific functional role, shows how defined elements of the IRES emerge from a compactly folded core and interact with the key ribosomal components that form the A, P and E sites, where tRNAs normally bind. Our results exemplify the molecular strategy for recruitment of an IRES and reveal the dynamic features necessary for internal initiation.Initiation of protein synthesis is an essential phase of protein synthesis and a key regulatory step in gene expression 1,2 . In eukaryotes, the canonical 5¢ cap-dependent pathway is facilitated and orchestrated by approximately 11 translation initiation factors. However, IRES RNAs can functionally substitute for initiation factors and facilitate the alternative pathway of internal initiation 3,4 . IRESs are present in 5¢ untranslated regions (UTRs) of many viral RNAs and are efficient tools to hijack the translational apparatus of the host during viral infection. They are also used by a subset of cellular messenger RNAsfor example, several proto-oncogenes [3][4][5] . In this context, they act as regulatory tools and are used to initiate translation during cellular stress or other periods when overall global translation is compromised.The molecular mechanisms of initiation by IRES RNAs are largely unknown. IRES RNAs fall into different classes that are distinguished by their structure and dependence on different sets of canonical initiation factors and IRES trans-acting factors 3-6 . The simplest mechanism of initiation is used by the intergenic IRESs of dicistroviruses, such as the cricket paralysis virus (CrPV). This family of IRESs does not require any initiation factor or even initiator tRNA in order to assemble elongation-competent 80S ribosomes 7-10 . According to biochemical studies, the IRES binds directly to the ribosomal 40S subunit and sets the translational reading frame by positioning the first codon into the ribosomal A site. This is highly unusual, because canonical initiation starts from the P site. Moreover, like the hepatitis C virus IRES 11 , the CrPV IRES actively manipulates the conformation of the translational machinery, suggesting that the IRES acts like an RNA-based translation factor 12 .A detailed knowledge of the CrPV IRES structure, especially in the ribosome-bound state, is a prerequisite for understanding the mechanism of internal initiation without initiation factors. The low resolution of the previous cryo-EM maps limit...

Elongation factor G (EF-G) catalyzes tRNA translocation on the ribosome. Here a cryo-EM reconstruction of the 70S*EF-G ribosomal complex at 7.3 A resolution and the crystal structure of EF-G-2*GTP, an EF-G homolog, at 2.2 A resolution are presented. EF-G-2*GTP is structurally distinct from previous EF-G structures, and in the context of the cryo-EM structure, the conformational changes are associated with ribosome binding and activation of the GTP binding pocket. The P loop and switch II approach A2660-A2662 in helix 95 of the 23S rRNA, indicating an important role for these conserved bases. Furthermore, the ordering of the functionally important switch I and II regions, which interact with the bound GTP, is dependent on interactions with the ribosome in the ratcheted conformation. Therefore, a network of interaction with the ribosome establishes the active GTP conformation of EF-G and thus facilitates GTP hydrolysis and tRNA translocation.

We have used single-particle reconstruction in cryo-electron microscopy to determine a structure of the Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF-Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin. This represents the state in the decoding process just after codon recognition by tRNA and the resulting GTP hydrolysis by EF-Tu, but before the release of EF-Tu from the ribosome. Progress in sample purification and image processing made it possible to reach a resolution of 6.4 Å . Secondary structure elements in tRNA, EF-Tu and the ribosome, and even GDP and kirromycin, could all be visualized directly. The structure reveals a complex conformational rearrangement of the tRNA in the A/T state and the interactions with the functionally important switch regions of EF-Tu crucial to GTP hydrolysis. Thus, the structure provides insights into the molecular mechanism of signalling codon recognition from the decoding centre of the 30S subunit to the GTPase centre of EF-Tu.

SUMMARY The extent to which bacterial ribosomes and the significantly larger eukaryotic ribosomes share the same mechanisms of ribosomal elongation is unknown. Here, we present sub-nanometer resolution cryo-electron microscopy maps of the mammalian 80S ribosome in the post-translocational state and in complex with the eukaryotic eEF1A•Val-tRNA•GMPPNP ternary complex, revealing significant differences in the elongation mechanism between bacteria and mammals. Surprisingly, and in contrast to bacterial ribosomes, a rotation of the small subunit around its long axis and orthogonal to the well-known intersubunit rotation distinguishes the post-translocational state from the classical pre-translocational state ribosome. We term this motion “subunit rolling”. Correspondingly, a mammalian decoding complex visualized in sub-states before and after codon recognition reveals structural distinctions from the bacterial system. These findings suggest how codon recognition leads to GTPase activation in the mammalian system and demonstrate that in mammalia subunit rolling occurs during tRNA selection.

Although the structural core of the ribosome is conserved in all kingdoms of life, eukaryotic ribosomes are significantly larger and more complex than their bacterial counterparts. The extent to which these differences influence the molecular mechanism of translation remains elusive. Multiparticle cryo-electron microscopy and single-molecule FRET investigations of the mammalian pre-translocation complex reveal spontaneous, large-scale conformational changes including an inter-subunit rotation of the ribosomal subunits. Through structurally related processes, tRNA substrates oscillate between classical and at least two distinct hybrid configurations facilitated by localized changes in their L-shaped fold. Hybrid states are favoured within the mammalian complex. However, classical tRNA positions can be restored by tRNA binding to the E site or by the eukaryotic-specific antibiotic and translocation inhibitor, cycloheximide. These findings reveal critical distinctions in the structural and energetic features of bacterial and mammalian ribosomes, providing a mechanistic basis for divergent translation regulation strategies and species-specific antibiotic action.

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Among cyclic nucleotide phosphodiesterases (PDEs), PDE6 is unique in serving as an effector enzyme in G protein-coupled signal transduction. In retinal rods and cones, PDE6 is membrane-bound and activated to hydrolyse its substrate, cGMP, by binding of two active G protein α-subunits (Gα*). To investigate the activation mechanism of mammalian rod PDE6, we have collected functional and structural data, and analysed them by reaction–diffusion simulations. Gα* titration of membrane-bound PDE6 reveals a strong functional asymmetry of the enzyme with respect to the affinity of Gα* for its two binding sites on membrane-bound PDE6 and the enzymatic activity of the intermediary 1 : 1 Gα* · PDE6 complex. Employing cGMP and its 8-bromo analogue as substrates, we find that Gα* · PDE6 forms with high affinity but has virtually no cGMP hydrolytic activity. To fully activate PDE6, it takes a second copy of Gα* which binds with lower affinity, forming Gα* · PDE6 · Gα*. Reaction–diffusion simulations show that the functional asymmetry of membrane-bound PDE6 constitutes a coincidence switch and explains the lack of G protein-related noise in visual signal transduction. The high local concentration of Gα* generated by a light-activated rhodopsin molecule efficiently activates PDE6, whereas the low density of spontaneously activated Gα* fails to activate the effector enzyme.

The activation kinetics of constitutive and IFNg-stimulated 20S proteasomes obtained with homomeric (recPA28a, recPA28b) and heteromeric (recPA28ab) forms of recombinant 11S regulator PA28 was analysed by means of kinetic modelling.The activation curves obtained with increasing concentrations of the individual PA28 subunits (RecP28a/ RecP28b/RecP28a 1 RecP28b) exhibit biphasic characteristics which can be attributed to a low-level activation by PA28 monomers and full proteasome activation by assembled activator complexes. The dissociation constants do not reveal significant differences between the constitutive and the immunoproteasome. Intriguingly, the affinity of the proteasome towards the recPA28ab complex is about two orders of magnitude higher than towards the homomeric PA28a and PA28b complexes.Striking similarities can been revealed in the way how PA28 mediates the kinetics of latent proteasomes with respect to three different fluorogenic peptides probing the chymotrypsin-like, trypsin-like and peptidylglutamylpeptide hydrolyzing like activity: (a) positive cooperativity disappears as indicated by a lack of sigmoid initial parts of the kinetic curves, (b) substrate affinity is increased, whereby (c), the maximal activity remains virtually constant. As these kinetic features are independent of the peptide substrates, we conclude that PA28 exerts its activating influence on the proteasome by enhancing the uptake (and release) of shorter peptides.Keywords: 20S proteasome; fluorogenic peptides; kinetic modelling; PA28 activator.The eukaryotic 20S proteasome represents a new class of N-terminal threonine hydrolases containing six compartmentalized active sites which are hidden in the internal catalytic chamber [1]. The 20S complex is composed of 28 subunits, arranged in an a 7 b 7 b 7 a 7 stoichiometry. The two external heptameric a-type rings serve as interacting interface for the association of regulatory proteins whereas each of the two internal b-type rings harbours three different proteolytically active sites, provided by the amino-terminal residues of three constitutive subunits b1 (X/d), b2 (Z/MC14) and b5 (Y/MB1) [2]. In lymphatic tissues or upon treatment with interferon-g, these constitutive subunits are replaced by the homologous active site subunits ib1 (LMP2), ib2 (MECL-1) and ib5 (LMP7) to form what are known as immunoproteasomes [3]. The replacements as well as the association of 20S proteasomes with several modulators, such as proteasome activator PA28, have been assumed to induce conformational changes which may affect the catalytic sites of 20S proteasomes [4±7].The proteasome activator PA28 was initially identified from its ability to enhance the turnover of standard small fluorogenic peptide substrates [8,9]. The tissue derived PA28 complex is a 180±200-kDa heteromeric protein complex composed of two IFN-g inducible closely related subunits PA28a and PA28b with molecular masses of 28.6 kDa and 27 kDa, respectively [10±12]. From titration and coimmunoprecipitation experiments it was conclude...