The archaeal ribosomal stalk complex has been shown to have an apparently conserved functional structure with eukaryotic pentameric stalk complex; it provides access to eukaryotic elongation factors at levels comparable to that of the eukaryotic stalk. The crystal structure of the archaeal heptameric (P0(P1) 2 (P1) 2 (P1) 2 ) stalk complex shows that the rRNA anchor protein P0 consists of an N-terminal rRNA-anchoring domain followed by three separated spine helices on which three P1 dimers bind. Based on the structure, we have generated P0 mutants depleted of any binding site(s) for P1 dimer(s). Factordependent GTPase assay of such mutants suggested that the first P1 dimer has higher activity than the others. Furthermore, we constructed a model of the archaeal 50 S with stalk complex by superposing the rRNA-anchoring domain of P0 on the archaeal 50 S. This model indicates that the C termini of P1 dimers where translation factors bind are all localized to the region between the stalk base of the 50 S and P0 spine helices. Together with the mutational experiments we infer that the functional significance of multiple copies of P1 is in creating a factor pool within a limited space near the stalk base of the ribosome.
The molecular mechanisms of translation termination and mRNA surveillance in archaea remain unclear. In eukaryotes, eRF3 and HBS1, which are homologous to the tRNA carrier GTPase EF1α, respectively bind eRF1 and Pelota to decipher stop codons or to facilitate mRNA surveillance. However, genome-wide searches of archaea have failed to detect any orthologs to both GTPases. Here, we report the crystal structure of aRF1 from an archaeon, Aeropyrum pernix, and present strong evidence that the authentic archaeal EF1α acts as a carrier GTPase for aRF1 and for aPelota. The binding interface residues between aRF1 and aEF1α predicted from aRF1·aEF1α·GTP ternary structure model were confirmed by in vivo functional assays. The aRF1/eRF1 structural domain with GGQ motif, which corresponds to the CCA arm of tRNA, contacts with all three structural domains of aEF1α showing striking tRNA mimicry of aRF1/eRF1 and its GTPase-mediated catalysis for stop codon decoding. The multiple binding capacity of archaeal EF1α explains the absence of GTPase orthologs for eRF3 and HBS1 in archaea species and suggests that universal molecular mechanisms underlie translational elongation and termination, and mRNA surveillance pathways.genetic code | protein quality control | tRNA mimicy
The FeoB family proteins are widely distributed prokaryotic membrane proteins involved in Fe(2+) uptake. FeoB consists of N-terminal cytosolic and C-terminal transmembrane domains. The N-terminal region of the cytosolic domain is homologous to small GTPase (G) proteins and is considered to regulate Fe(2+) uptake. The spacer region connecting the G and TM domains reportedly functions as a GDP dissociation inhibitor (GDI)-like domain that stabilizes the GDP-binding state. However, the function of the G and GDI-like domains in iron uptake remains unclear. Here, we report the structural and functional analyses of the FeoB cytosolic domain from Thermotoga maritima. The structure-based mutational analysis indicated that the interaction between the G and GDI-like domains is important for both the GDI and Fe(2+) uptake activities. On the basis of these results, we propose a regulatory mechanism of Fe(2+) uptake.
Walker-Warburg syndrome (WWS) is a congenital muscular dystrophy associated with neuronal migration disorder and structural eye abnormalities. The mutations in the O-mannosyltransferase 1 gene (POMT1) were identified recently in 20% of patients with WWS. The authors report on a patient with WWS and a novel POMT1 mutation. Their patient expressed alpha-dystroglycan (alpha-DG) core protein, but fully glycosylated alpha-DG antibody epitopes were absent, associated with the loss of laminin-binding activity.
Cycloheximide is one of the antibiotics that inhibit protein synthesis in most eukaryotic cells. We have found that a yeast, Candida maltosa, is resistant to the drug because it possesses a cycloheximide-resistant ribosome, and we have isolated the gene responsible for this. In this study, we sequenced this gene and found that the gene encodes a protein homologous to the L41 ribosomal protein of Saccharomyces cerevisiae, whose amino acid sequence has already been reported. Two genes for L41 protein, named L41a and L41b, independently present in the genome of S. cerevisiae, were isolated. L41-related genes were also isolated from a few other yeast species. Each of these genes has an intron at the same site of the open reading frame. Comparison of their deduced amino acid sequences and their ability to confer cycloheximide resistance to S. cerevisiae, when introduced in a high-copy-number plasmid, suggested that the 56th amino acid residue of the L41 protein determines the sensitivity of the ribosome to cycloheximide; the amino acid is glutamine in the resistant ribosome, whereas that in the sensitive ribosome is proline. This was confirmed by constructing a cycloheximide-resistant strain of S. cerevisiae having a disrupted L41a gene and an L41b gene with a substitution of the glutamine codon for the proline codon.
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