Localization of eukaryote-specific ribosomal proteins in a 5.5-Å cryo-EM map of the 80S eukaryotic ribosome

Abstract

Protein synthesis in all living organisms occurs on ribonucleoprotein particles, called ribosomes. Despite the universality of this process, eukaryotic ribosomes are significantly larger in size than their bacterial counterparts due in part to the presence of 80 r proteins rather than 54 in bacteria. Using cryoelectron microscopy reconstructions of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution, together with a 6.1-Å map of a translating Saccharomyces cerevisiae 80S ribosome, we have localized and modeled 74∕80 (92.5%) of the ribosomal proteins, encompassing 12 archaeal/eukaryote-specific small subunit proteins as well as the complete complement of the ribosomal proteins of the eukaryotic large subunit. Near-complete atomic models of the 80S ribosome provide insights into the structure, function, and evolution of the eukaryotic translational apparatus.homology modeling | RNA | translation | flexible fitting | molecular dynamics P rotein synthesis occurs on large macromolecular complexes, called ribosomes (1). Ribosomes are composed of two subunits, both of which are built from protein and RNA. Bacterial ribosomes, for example, in Escherichia coli, contain a small subunit composed of one 16S rRNA and 21 ribosomal proteins (r proteins), and a large subunit containing 5S and 23S rRNAs and 33 r proteins. In contrast, eukaryotic ribosomes are much larger and more complex, containing additional RNA in the form of so-called expansion segments (ES) as well as many additional r proteins and r-protein extensions. The additional r proteins present in eukaryotic ribosomes are likely to reflect the increased complexity of translation regulation in eukaryotic cells (2-5). Moreover, many of these eukaryote-specific components have been associated with human disorders (4). Thus, structural insight into the localization of these elements will be important to furthering our understanding of eukaryotic translation regulation as well as disease.Compared with the ∼54 r proteins of the bacterial ribosome, plant and fungal 80S ribosomes contain ∼80 r proteins (see Table S1 for r-protein nomenclature). Crystal structures have revealed the location of each small and large subunit r protein within bacterial ribosomes (6-12) as well as the r proteins within the archaeal large ribosomal subunit (13,14). In contrast, the localization of ribosomal proteins within eukaryotic 80S ribosomes has come mainly from early studies using immuno-EM and cross-linking approaches (see, for example, refs. 15-18). Moreover, the first molecular models for the eukaryotic ribosome were built at 15-Å resolution by docking the structures of the bacterial small 30S subunit (6) and archaeal large 50S subunit (13), thus only identifying the location of a total of 46 eukaryotic r proteins with bacterial or archaeal homologues (19). Recently, cryo-EM reconstructions of plant and fungal 80S ribosomes have led to the localization of three eukaryote-specific r proteins: RACK1 (20) and S19e (21) in the small subunit and L30e in the large subunit...

60S ribosome biogenesis requires rotation of the 5S ribonucleoprotein particle

“…8). This revealed that the position of Mrt4 is indeed very similar to that of P0 in the mature yeast 80S ribosome 30 . Moreover, Mrt4 binds close to Nog1 (see below) and the r-protein L11 (Fig.…”

“…R-proteins lacking in the template structure were taken from a cryo-EM model of the yeast ribosome: L1 and L11 (PDB ID: 3IZS) 30 . For each r-protein, the presence of the corresponding density was checked and missing r-proteins were removed from the model accordingly.…”

Defining the limits and reliability of rigid-body fitting in cryo-EM maps using multi-scale image pyramids

“…The ribosome is an excellent case study as it contains many chains of various sizes and types. (Armache et al, 2010) 5.5 4V7E 4.2 88 2620 (Budkevich et al, 2014) 6.9 4UJE 4.5 83 2845 (Aylett et al, 2015) 6.5 4UER 1.2 39 5591 (Anger et al, 2013) 6.0 4V6W 4.0 86 5976 (Svidritskly et al, 2014) 6.2 3J77 3.5 83…”

Activities of the peptidyl transferase center of ribosomes lacking protein L27

“…Furthermore, the α-amino group of L27 was recently suggested to take part in proton transfer during the PT reaction, assisting the 5 ′ -phosphate oxygen of the A-site A76 in the deprotonation of the nucleophile in the transition state (Polikanov et al 2014). Although L27 is not present in Archaea and Eukaryotes, another ribosomal protein, the archaeal L10e and its eukaryotic homolog RPL10, extends a loop in the same position as the L27 tail (Armache et al 2010); mutations in this conserved loop are lethal (Hofer et al 2007). Validation of the role of L27 in the PT reaction for all or perhaps only certain incoming amino acids would, however, require detailed biochemical and kinetic studies.…”