Robert A. Grassucci

Structure of the signal recognition particle interacting with the elongation-arrested ribosome

Cotranslational translocation of proteins across or into membranes is a vital process in all kingdoms of life. It requires that the translating ribosome be targeted to the membrane by the signal recognition particle (SRP), an evolutionarily conserved ribonucleoprotein particle. SRP recognizes signal sequences of nascent protein chains emerging from the ribosome. Subsequent binding of SRP leads to a pause in peptide elongation and to the ribosome docking to the membrane-bound SRP receptor. Here we present the structure of a targeting complex consisting of mammalian SRP bound to an active 80S ribosome carrying a signal sequence. This structure, solved to 12u2009Å by cryo-electron microscopy, enables us to generate a molecular model of SRP in its functional conformation. The model shows how the S domain of SRP contacts the large ribosomal subunit at the nascent chain exit site to bind the signal sequence, and that the Alu domain reaches into the elongation-factor-binding site of the ribosome, explaining its elongation arrest activity.

EF-G-dependent GTP hydrolysis induces translocation accompanied by large conformational changes in the 70S ribosome

Cryo-electron microscopy has been used to visualize elongation factor G (EF-G) on the 70S ribosome in GDP and GTP states. GTP hydrolysis is required for binding of all the domains of EF-G to the pretranslocational complex and for the completion of translocation. In addition, large conformational changes have been identified in the ribosome. The head of the 30S subunit shifts toward the L1 protein side, and the L7/L12 stalk becomes bifurcated upon EF-G binding. Upon GTP hydrolysis, the bifurcation is reversed and an arc-like connection is formed between the base of the stalk and EF-G.

The Polypeptide Tunnel System in the Ribosome and Its Gating in Erythromycin Resistance Mutants of L4 and L22

Variations in the inner ribosomal landscape determining the topology of nascent protein transport have been studied by three-dimensional cryo-electron microscopy of erythromycin-resistant Escherichia coli 70S ribosomes. Significant differences in the mouth of the 50S subunit tunnel system visualized in the present study support a simple steric-hindrance explanation for the action of the drug. Examination of ribosomes in different functional states suggests that opening and closing of the main tunnel are dynamic features of the large subunit, possibly accompanied by changes in the L7/L12 stalk region. The existence and dynamic behavior of side tunnels suggest that ribosomal proteins L4 and L22 might be involved in the regulation of a multiple exit system facilitating cotranslational processing (or folding or directing) of nascent proteins.

Locations of Calmodulin and FK506-binding Protein on the Three-dimensional Architecture of the Skeletal Muscle Ryanodine Receptor

Isolated skeletal muscle ryanodine receptors (RyRs) complexed with the modulatory ligands, calmodulin (CaM) or 12-kDa FK506-binding protein (FKBP12), have been characterized by electron cryomicroscopy and three-dimensional reconstruction. RyRs are composed of 4 large subunits (molecular mass 565 kDa) that assemble to form a 4-fold symmetric complex that, architecturally, comprises two major substructures, a large (≈80% of the total mass) cytoplasmic assembly and a smaller transmembrane assembly. Both CaM and FKBP12 bind to the cytoplasmic assembly at sites that are 10 and 12 nm, respectively, from the putative entrance to the transmembrane ion channel. FKBP12 binds along the edge of the square-shaped cytoplasmic assembly near the face that interacts in vivo with the sarcolemma/transverse tubule membrane system, whereas CaM binds within a cleft that faces the junctional face of the sarcoplasmic reticulum membrane at the triad junction. Both ligands interact with a domain that connects directly to a cytoplasmic extension of the transmembrane assembly of the receptor, and thus might cause structural changes in the domain which in turn modulate channel gating.

Three‐dimensional cryo‐electron microscopy localization of EF2 in the Saccharomyces cerevisiae 80S ribosome at 17.5 Å resolution

Using a sordarin derivative, an antifungal drug, it was possible to determine the structure of a eukaryotic ribosome·EF2 complex at 17.5 Å resolution by three‐dimensional (3D) cryo‐electron microscopy. EF2 is directly visible in the 3D map and the overall arrangement of the complex from Saccharomyces cerevisiae corresponds to that previously seen in Escherichia coli. However, pronounced differences were found in two prominent regions. First, in the yeast system the interaction between the elongation factor and the stalk region of the large subunit is much more extensive. Secondly, domain IV of EF2 contains additional mass that appears to interact with the head of the 40S subunit and the region of the main bridge of the 60S subunit. The shape and position of domain IV of EF2 suggest that it might interact directly with P‐site‐bound tRNA.

Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA.

Dynamic changes in secondary structure of the 16S rRNA during the decoding of mRNA are visualized by three‐dimensional cryo‐electron microscopy of the 70S ribosome. Thermodynamically unstable base pairing of the 912–910 (CUC) nucleotides of the 16S RNA with two adjacent complementary regions at nucleotides 885–887 (GGG) and 888–890 (GAG) was stabilized in either of the two states by point mutations at positions 912 (C912G) and 885 (G885U). A wave of rearrangements can be traced arising from the switch in the three base pairs and involving functionally important regions in both subunits of the ribosome. This significantly affects the topography of the A‐site tRNA‐binding region on the 30S subunit and thereby explains changes in tRNA affinity for the ribosome and fidelity of decoding mRNA.

Effect of buffer conditions on the position of tRNA on the 70 S ribosome as visualized by cryoelectron microscopy.

The effect of buffer conditions on the binding position of tRNA on the Escherichia coli 70 S ribosome have been studied by means of three-dimensional (3D) cryoelectron microscopy. Either deacylated tRNAf Met or fMet-tRNAf Met were bound to the 70 S ribosomes, which were programmed with a 46-nucleotide mRNA having AUG codon in the middle, under two different buffer conditions (conventional buffer: containing Tris and higher Mg2+ concentration [10–15 mm]; and polyamine buffer: containing Hepes, lower Mg2+ concentration [6 mm], and polyamines). Difference maps, obtained by subtracting 3D maps of naked control ribosome in the corresponding buffer from the 3D maps of tRNA·ribosome complexes, reveal the distinct locations of tRNA on the ribosome. The position of deacylated tRNAf Metdepends on the buffer condition used, whereas that of fMet-tRNAf Met remains the same in both buffer conditions. The acylated tRNA binds in the classical P site, whereas deacylated tRNA binds mostly in an intermediate P/E position under the conventional buffer condition and mostly in the position corresponding to the classical P site, i.e. in the P/P state, under the polyamine buffer conditions.

Structure of the mammalian ribosomal pre-termination complex associated with eRF1•eRF3•GDPNP

Eukaryotic translation termination results from the complex functional interplay between two release factors, eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, we present a cryo-electron microscopy structure of pre-termination complexes associated with eRF1•eRF3•GDPNP at 9.7 -Å resolution, which corresponds to the initial pre-GTP hydrolysis stage of factor attachment and stop codon recognition. It reveals the ribosomal positions of eRFs and provides insights into the mechanisms of stop codon recognition and triggering of eRF3’s GTPase activity.

Direct three-dimensional localization and positive identification of RNA helices within the ribosome by means of genetic tagging and cyro-electron microscopy

BACKGROUNDnRibosomes are complex macromolecular machines that perform the translation of the genetic message. Cryo-electron microscopic (cryo-EM) maps of the Escherichia coli 70S ribosome are approaching a resolution of 10 A and X-ray maps of the 30S and 50S subunits are now available at 5 A. These maps show a lot of details about the inner architecture of the ribosome and ribosomal RNA helices are clearly visible. However, in the absence of further biological information, even at the higher resolution of the X-ray maps many rRNA helices can be placed only tentatively. Here we show that genetic tagging in combination with cryo-EM can place and orient double-stranded RNA helices with high accuracy.nnnRESULTSnA tRNA sequence inserted into the E. coli 23S ribosomal RNA gene, at one of the points of sequence expansion in eukaryotic ribosomes, is visible in the cryo-EM map as a peripheral foot structure. By tracing its acceptor-stem end, the location of helix 63 in domain IV and helix 98 in domain VI of the 50S subunit could be precisely determined.nnnCONCLUSIONSnOur study demonstrates for the first time that features of a three-dimensional cryo-EM map of an asymmetric macromolecular complex can be interpreted in terms of secondary and primary structure. Using the identified helices as a starting point, it is possible to model and interpret, in molecular terms, a larger portion of the ribosome. Our results might be also useful in interpreting and refining the current X-ray maps.

The group II intron ribonucleoprotein precursor is a large, loosely packed structure

Group II self-splicing introns are phylogenetically diverse retroelements that are widely held to be the ancestors of spliceosomal introns and retrotransposons that insert into DNA. Folding of group II intron RNA is often guided by an intron-encoded protein to form a catalytically active ribonucleoprotein (RNP) complex that plays a key role in the activity of the intron. To date, possible structural differences between the intron RNP in its precursor and spliced forms remain unexplored. In this work, we have trapped the native Lactococcus lactis group II intron RNP complex in its precursor form, by deleting the adenosine nucleophile that initiates splicing. Sedimentation velocity, size-exclusion chromatography and cryo-electron microscopy provide the first glimpse of the intron RNP precursor as a large, loosely packed structure. The dimensions contrast with those of compact spliced introns, implying that the RNP undergoes a dramatic conformational change to achieve the catalytically active state.