Christian M.T. Spahn

Structure of the 80S Ribosome from Saccharomyces cerevisiae—tRNA-Ribosome and Subunit-Subunit Interactions

A cryo-EM reconstruction of the translating yeast 80S ribosome was analyzed. Computationally separated rRNA and protein densities were used for docking of appropriately modified rRNA models and homology models of yeast ribosomal proteins. The core of the ribosome shows a remarkable degree of conservation. However, some significant differences in functionally important regions and dramatic changes in the periphery due to expansion segments and additional ribosomal proteins are evident. As in the bacterial ribosome, bridges between the subunits are mainly formed by RNA contacts. Four new bridges are present at the periphery. The position of the P site tRNA coincides precisely with its prokaryotic counterpart, with mainly rRNA contributing to its molecular environment. This analysis presents an exhaustive inventory of an eukaryotic ribosome at the molecular level.

Architecture of the Protein-Conducting Channel Associated with the Translating 80S Ribosome

In vitro assembled yeast ribosome-nascent chain complexes (RNCs) containing a signal sequence in the nascent chain were immunopurified and reconstituted with the purified protein-conducting channel (PCC) of yeast endoplasmic reticulum, the Sec61 complex. A cryo-EM reconstruction of the RNC-Sec61 complex at 15.4 A resolution shows a tRNA in the P site. Distinct rRNA elements and proteins of the large ribosomal subunit form four connections with the PCC across a gap of about 10-20 A. Binding of the PCC influences the position of the highly dynamic rRNA expansion segment 27. The RNC-bound Sec61 complex has a compact appearance and was estimated to be a trimer. We propose a binary model of cotranslational translocation entailing only two basic functional states of the translating ribosome-channel complex.

Solution structure of the E. coli 70S ribosome at 11.5 A resolution.

Over 73,000 projections of the E. coli ribosome bound with formyl-methionyl initiator tRNAf(Met) were used to obtain an 11.5 A cryo-electron microscopy map of the complex. This map allows identification of RNA helices, peripheral proteins, and intersubunit bridges. Comparison of double-stranded RNA regions and positions of proteins identified in both cryo-EM and X-ray maps indicates good overall agreement but points to rearrangements of ribosomal components required for the subunit association. Fitting of known components of the 50S stalk base region into the map defines the architecture of the GTPase-associated center and reveals a major change in the orientation of the alpha-sarcin-ricin loop. Analysis of the bridging connections between the subunits provides insight into the dynamic signaling mechanism between the ribosomal subunits.

Cryo-EM Visualization of a Viral Internal Ribosome Entry Site Bound to Human Ribosomes: The IRES Functions as an RNA-Based Translation Factor

Internal initiation of protein synthesis in eukaryotes is accomplished by recruitment of ribosomes to structured internal ribosome entry sites (IRESs), which are located in certain viral and cellular messenger RNAs. An IRES element in cricket paralysis virus (CrPV) can directly assemble 80S ribosomes in the absence of canonical initiation factors and initiator tRNA. Here we present cryo-EM structures of the CrPV IRES bound to the human ribosomal 40S subunit and to the 80S ribosome. The CrPV IRES adopts a defined, elongate structure within the ribosomal intersubunit space and forms specific contacts with components of the ribosomal A, P, and E sites. Conformational changes in the ribosome as well as within the IRES itself show that CrPV IRES actively manipulates the ribosome. CrPV-like IRES elements seem to act as RNA-based translation factors.

A new system for naming ribosomal proteins

A system for naming ribosomal proteins is described that the authors intend to use in the future. They urge others to adopt it. The objective is to eliminate the confusion caused by the assignment of identical names to ribosomal proteins from different species that are unrelated in structure and function. In the system proposed here, homologous ribosomal proteins are assigned the same name, regardless of species. It is designed so that new names are similar enough to old names to be easily recognized, but are written in a format that unambiguously identifies them as new system names.

Ribosomal protein L2 is involved in the association of the ribosomal subunits, tRNA binding to A and P sites and peptidyl transfer

Ribosomal proteins L2, L3 and L4, together with the 23S RNA, are the main candidates for catalyzing peptide bond formation on the 50S subunit. That L2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50S particles either lacking L2 or harboring a mutated L2. L2 does not play a dominant role in the assembly of the 50S subunit or in the fixation of the 3′‐ends of the tRNAs at the peptidyl‐transferase center. However, it is absolutely required for the association of 30S and 50S subunits and is strongly involved in tRNA binding to both A and P sites, possibly at the elbow region of the tRNAs. Furthermore, while the conserved histidyl residue 229 is extremely important for peptidyl‐transferase activity, it is apparently not involved in other measured functions. None of the other mutagenized amino acids (H14, D83, S177, D228, H231) showed this strong and exclusive participation in peptide bond formation. These results are used to examine critically the proposed direct involvement of His229 in catalysis of peptide synthesis.

Localization of the Ribosomal Protection Protein Tet(O) on the Ribosome and the Mechanism of Tetracycline Resistance

Tet(O) belongs to a class of ribosomal protection proteins that mediate tetracycline resistance. It is a G protein that shows significant sequence similarity to elongation factor EF-G. Here we present a cryo-electron microscopic reconstruction, at 16 A resolution, of its complex with the E. coli 70S ribosome. Tet(O) was bound in the presence of a noncleavable GTP analog to programmed ribosomal complexes carrying fMet-tRNA in the P site. Tet(O) is directly visible as a mass close to the A-site region, similar in shape and binding position to EF-G. However, there are important differences. One of them is the different location of the tip of domain IV, which in the Tet(O) case, does not overlap with the ribosomal A site but is directly adjacent to the primary tetracycline binding site. Our findings give insights into the mechanism of tetracycline resistance.

EF-G and EF4: translocation and back-translocation on the bacterial ribosome.

Ribosomes translate the codon sequence of an mRNA into the amino acid sequence of the corresponding protein. One of the most crucial events is the translocation reaction, which involves movement of both the mRNA and the attached tRNAs by one codon length and is catalysed by the GTPase elongation factor G (EF-G). Interestingly, recent studies have identified a structurally related GTPase, EF4, that catalyses movement of the tRNA2–mRNA complex in the opposite direction when the ribosome stalls, which is known as back-translocation. In this Review, we describe recent insights into the mechanistic basis of both translocation and back-translocation.

The ribosome and the mechanism of protein synthesis

In virtually all forms of life on earth, proteins in each cell are made according to a genetic blueprint, in the form of DNA. The translation of copies of this genetic blueprint (in the form of messenger RNA) into polypeptides is performed on the ribosome, a highly complex molecular machine composed of RNAs and proteins. To this end, special adaptor molecules called transfer RNAs are lined up by the ribosome in the sequence dictated by the genetic code, such that the amino acids carried by these molecules can be linked into a polypeptide. Several cofactors are involved in these processes, some of which require energy freed up by GTP hydrolysis. Although the ribosome was discovered more than 50 years ago, its structure has only been solved recently by X-ray crystallography. Another technique, cryo-electron microscopy, is starting to contribute toward our understanding of the ribosomes function, by portraying its conformational changes and binding interactions with the cofactors and tRNA.

Multiparticle cryo-EM of ribosomes.

As the resolution of cryo-EM reconstructions has improved to the subnanometer range, conformational and compositional heterogeneity have become increasing problems in cryo-EM, limiting the resolution of reconstructions. Since further purification is not feasible, the presence of several conformational states of ribosomal complexes in thermodynamic equilibrium requires methods for separating these states in silico. We describe a procedure for generating subnanometer resolution cryo-EM structures from large sets of projection images of ribosomal complexes. The incremental K-means-like method of unsupervised 3D sorting discussed here allows separation of classes in the dataset by exploiting intrinsic divisions in the data. The classification procedure is described in detail and its effectiveness is illustrated using current examples from our work. Through a good separation of conformational modes, higher resolution reconstructions can be calculated. This increases information gained from single states, while exploiting the coexistence of multiple states to gather comprehensive mechanistic insight into biological processes like ribosomal translocation.