A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits

The large ribosomal subunit catalyzes peptide bond formation during protein synthesis. Its peptidyl transferase activity has often been studied using a 'fragment assay' that depends on high concentrations of methanol or ethanol. Here we describe a version of this assay that does not require alcohol and use it to show, both crystallographically and biochemically, that crystals of the large ribosomal subunits from Haloarcula marismortui are enzymatically active. Addition of these crystals to solutions containing substrates results in formation of products, which ceases when crystals are removed. When substrates are diffused into large subunit crystals, the subsequent structure shows that products have formed. The CC-puromycin-peptide product is found bound to the A-site and the deacylated CCA is bound to the P-site, with its 3prime prime or minute OH near N3 A2486 (Escherichia coli A2451). Thus, this structure represents a state that occurs after peptide bond formation but before the hybrid state of protein synthesis.     

Structures of deacylated tRNA mimics bound to the E site of the large ribosomal subunit

During translation, tRNAs cycle through three binding sites on the ribosome: the A, the P, and the E sites. We have determined the structures of complexes between the Haloarcula marismortui large ribosomal subunit and two different E site substrates: a deacylated tRNA acceptor stem minihelix and a CCA-acceptor end. Both of these tRNA mimics contain analogs of adenosine 76, the component responsible for a large proportion of E site binding affinity. They bind in the center of the loop-extension of protein L44e, and make specific contacts with both L44e and 23S rRNA including bases that are conserved in all three kingdoms of life. These contacts are consistent with the footprinting, protection, and cross-linking data that have identified the E site biochemically. These structures explain the specificity of the E site for deacylated tRNAs, as it is too small to accommodate any relevant aminoacyl-tRNA. The orientation of the minihelix suggests that it may mimic the P/E hybrid state. It appears that the E site on the 50S subunit was formed by only RNA in the last common ancestor of the three kingdoms, since the proteins at the E sites of H. marismortui and Deinucoccus radiodurans large subunits are not homologous.     

IL-11 and IL-11Rα immunolocalisation at primate implantation sites supports a role for IL-11 in placentation and fetal development

Embryo implantation, endometrial stromal cell decidualization and formation of a functional placenta are critical processes in the establishment and maintenance of pregnancy. Interleukin (IL)-11 signalling is essential for adequate decidualization in the mouse uterus and IL-11 promotes decidualization in the human. IL-11 action is mediated via binding to the specific IL-11 receptor α (IL-11Rα). The present study examined immunoreactive IL-11 and IL-11Rα in cycling rhesus monkey endometrium, at implantation sites in cynomolgus and rhesus monkeys and in human first trimester decidua and defined distinct spatial and temporal patterns. In cycling rhesus monkey endometrium, IL-11 and IL-11Rα increased in both basalis and functionalis regions during the secretory compared with the proliferative phase, with changing cellular locations in luminal and glandular epithelium and stroma. The patterns were similar overall to those previously described in human endometrium. Differences were seen in immunostaining during implantation in cynomologus and rhesus monkey. In the cynomolgus, very little staining for IL-11 or IL-11Rα was seen in syncytio- and cyto-trophoblast cells in the villi between days 12 and 150 of pregnancy although there was moderate staining in cytotrophoblast in the shell between days 12 and 17 and in subpopulations of cytotrophoblast cells invading the arteries at day 17. By contrast in the rhesus monkey between days 24 and 35 of pregnancy and in human first trimester placenta, cyto- and syncytio-trophoblast in the villi but not cytotrophoblast in the shell were positively stained. The most intense staining for both IL-11 and IL-11Rα was present within the decidua in the maternal component of implantation sites in all three primates but moderate staining was also present in maternal vascular smooth muscle and glands perivascular cells and epithelial plaques. These results are consistent with a role for IL-11 both during decidualization and placentation in primates.     

Mitochondrial DNA variability in the Canary Islands honeybees (Apis mellifera L.)

The mitochondrial DNA (mtDNA) of individuals from 79 colonies of Apis mellifera from five Canary Islands was studied using the Dra I test based on the restriction of PCR products of the tRNAleu–COII intergenic region. Five haplotypes of the African (A) lineage and one of the west European (C) lineage were found. The haplotypes A14 and A15 are described for the first time. These haplotypes have a new P sequence named P₁. The wide distribution and high frequency of haplotype A15 suggest that it is characteristic of the Canarian Archipelago. Sources of haplotype variability of honeybee mtDNA in the Canary Islands (waves of colonization from Africa, queen importations, habitat diversification) are discussed.