Structures of the human eIF4E homologous protein, h4EHP, in its m7GTP-bound and unliganded forms

All eukaryotic cellular mRNAs contain a 5' m(7)GpppN cap. In addition to conferring stability to the mRNA, the cap is required for pre-mRNA splicing, nuclear export and translation by providing an anchor point for protein binding. In translation, the interaction between the cap and the eukaryotic initiation factor 4E (eIF4E) is important in the recruitment of the mRNAs to the ribosome. Human 4EHP (h4EHP) is a homologue of eIF4E. Like eIF4E it is able to bind the cap but it appears to play a different cellular role, possibly being involved in the fine-tuning of protein expression levels. Here we use X-ray crystallography and isothermal titration calorimetry (ITC) to investigate further the binding of cap analogues and peptides to h4EHP. m(7)GTP binds to 4EHP 200-fold more weakly than it does to eIF4E with the guanine base sandwiched by a tyrosine and a tryptophan instead of two tryptophan residues as seen in eIF4E. The tyrosine resides on a loop that is longer in h4EHP than in eIF4E. The consequent conformational difference between the proteins allows the tyrosine to mimic the six-membered ring of the tryptophan in eIF4E and adopt an orientation that is similar to that seen for equivalent residues in other non-homologous cap-binding proteins. In the absence of ligand the binding site is incompletely formed with one of the aromatic residues being disordered and the side-chain of the other adopting a novel conformation. A peptide derived from the eIF4E inhibitory protein, 4E-BP1 binds h4EHP 100-fold less strongly than eIF4E but in a similar manner. Overall the data, combined with sequence analyses of 4EHP from evolutionary diverse species, strongly support the hypothesis that 4EHP plays a physiological role utilizing both cap-binding and protein-binding functions but which is distinct from eIF4E.     

Computational determination of refractive index distribution in the crystalline cones of the compound eye of Antarctic krill (Euphausia superba)

In order to understand how a compound eye channels light to the retina and forms an image, one needs to know the refractive index distribution in the crystalline cones. Direct measurements of the refractive indices require sections of fresh, unfixed tissue and the use of an interference microscope, but frequently neither is available. Using the eye of the Antarctic krill Euphausia superba (the main food of baleen whales) we developed a computational method to predict a likely refractive index distribution non-invasively from sections of fixed material without the need of an interference microscope. We used a computer model of the eye and calculated the most realistic spatial distribution of the refractive index gradient in the crystalline cone that would enable the eye to produce a sharp image on the retina. The animals are known to see well and on the basis of our computations we predict that for the eyes of the adult a maximum refractive index of 1.45-1.50 in the centre of the cone yields a better angular sensitivity and light absorption in a target receptor of the retina than if N(max) were 1.55. In juveniles with a narrower spatial separation between dioptric structures and retina, however, an N(max) of 1.50-1.55 gives a superior result. Our method to determine the most likely refractive index distribution in the cone without the need of fresh material and an interference microscope could be useful in the study of other invertebrate eyes that are known to possess good resolving power, but for a variety of reasons are not suitable for or will not permit direct refractive index measurements of their dioptric tissues to be taken.     

Role of eIF3a (eIF3 p170) in intestinal cell differentiation and its association with early development

Eukaryotic initiation factor 3a (eIF3a) has been suggested to play a regulatory role in mRNA translation. Decreased eIF3a expression has been observed in differentiated cells while higher levels have been observed in cancer cells. However, whether eIF3a plays any role in differentiation and development is currently unknown. Here, we investigated eIF3a expression during mouse development and its role in differentiation of colon epithelial cells. We found that eIF3a expression was higher in fetal tissues compared with postnatal ones. Its expression in intestine, stomach, and lung abruptly stopped on the 18th day in gestation but persisted in liver, kidney, and heart throughout the postnatal stage at decreased levels. Similarly, eIF3a expression in colon cancer cell lines, HT-29 and Caco-2, drastically decreased prior to differentiation. Enforced eIF3a expression inhibited while knocking it down using small interference RNA promoted Caco-2 differentiation. Thus, eIF3a may play some roles in development and differentiation and that the decreased eIF3a expression may be a pre-requisite of intestinal epithelial cell differentiation.     

Integrating S-phase checkpoint signaling with trans-lesion synthesis of bulky DNA adducts

Bulky adducts are DNA lesions generated in response to environmental agents including benzo[a]pyrene (a combustion product) and solar ultraviolet radiation. Error-prone replication of adducted DNA can cause mutations, which may result in cancer. To minimize the detrimental effects of bulky adducts and other DNA lesions, S-phase checkpoint mechanisms sense DNA damage and integrate DNA repair with ongoing DNA replication. The essential protein kinase Chk1 mediates the S-phase checkpoint, inhibiting initiation of new DNA synthesis and promoting stabilization and recovery of stalled replication forks. Here we review the mechanisms by which Chk1 is activated in response to bulky adducts and potential mechanisms by which Chk1 signaling inhibits the initiation stage of DNA synthesis. Additionally, we discuss mechanisms by which Chk1 signaling facilitates bypass of bulky lesions by specialized Y-family DNA polymerases, thereby attenuating checkpoint signaling and allowing resumption of normal cell cycle progression.     

Effects of fighting decisions on formation and structure of dominance hierarchies

The characteristics of complex social systems often cannot be predicted from exploring individual agonistic interactions in isolation. In a series of models, this study linked the decision-making processes that govern dyadic interactions with the emergent properties of hierarchical structures in social groups. Contrary to the intuitive expectation that resolution of ambiguities in fighting between closely matched opponents effectively promotes the formation of a social hierarchy, engagement in contests with opponents of dissimilar status lead to a faster emergence of hierarchical structures. The increased certainty of outcome in such asymmetric contests both reinforced the previously established dominance, and allowed for an indirect resolution of existing ambiguous relationships. High-return fights also resulted in a more rapid hierarchy formation, while escalating in fights decreased the costs of maintaining hierarchical relationships and increased their stability.     

Microscopical investigation of poly(3-hydroxybutyrate) granule formation in Azotobacter vinelandii

Poly(3-hydroxybutyrate) (PHB) granule formation in Azotobacter vinelandii was investigated by laser scanning fluorescence microscopy after staining the cells with Nilered and Baclight. Cells that had been starved for a carbon source for > or =3 days were almost free of PHB granules. Formation of visible PHB granules started within 1-2 h after transfer of the cells to a medium permissive for PHB accumulation. Fluorescent PHB granules at the early stages of formation were exclusively found in the cell periphery of the 2-3 mum ovoid-shaped cells. After 3 h of PHB accumulation or later, PHB granules were also found to be detached from the cell periphery. Our results indicate that PHB granule formation apparently begins at the inner site of the cytoplasmic membrane. This finding is different from previous assumptions that PHB granule formation occurs randomly in the cytoplasm of PHB-accumulating bacteria.     

Liquid medium and liquid overlays improve embryogenic tissue initiation in conifers

Somatic embryogenesis (SE) is expected to play an important role in increasing productivity, sustainability, and uniformity of future US forests. For commercial use, SE technology must work with a variety of genetically diverse trees. Initiation in loblolly pine (Pinus taeda L.), the main commercial US forest species, is often recalcitrant for desirable genotypes. Liquid initiation medium with no or low gelling agent or placement of the explant on gelled medium followed later by a liquid medium overlay during the initiation process increased initiation for loblolly pine and Norway spruce (Picea abies). Loblolly pine liquid medium required reduction of NAA from 2 mg/l in gelled medium to 0.3 mg/l in liquid medium. Once the NAA concentration was adjusted, loblolly pine initiation occurred in liquid medium with fully immersed megagametophytes, explants supported at the liquid medium surface, or on gelled medium overlaid with liquid medium. Liquid overlays (0.25 ml) consisting of medium with NAA reduced to 0.3 mg/l, 9 mg/l ABA and no gelling agent applied to explants on 2 ml of gelled medium provided excellent initiation results. Greatest initiation percentages occurred when the liquid overlay was applied 14 days after placement of the megagametophyte on gelled medium. Initiation increases ranged from +8.5% with high-value cross-pollinated seed sources to +6.5 to +9.9% with open-pollinated and often recalcitrant seed sources. Liquid medium addition allows rapid replenishment of nutrients and adjustment or change of pH, hormones, or other parameters without disturbing the tissue.     

An Alternative Role of RluD in the Fidelity of Translation Initiation in Escherichia coli

The fidelity of initiator tRNA (i-tRNA) selection in the ribosomal P-site is a key step in translation initiation. The highly conserved three consecutive G:C base pairs (3GC pairs) in the i-tRNA anticodon stem play a crucial role in its selective binding in the P-site. Mutations in the 3GC pairs (3GC mutant) render the i-tRNA inactive in initiation. Here, we show that a mutation (E265K) in the unique C-terminal tail domain of RluD, a large ribosomal subunit pseudouridine synthase, results in compromised fidelity of initiation and allows initiation with the 3GC mutant i-tRNA. RluD modifies the uridine residues in H69 to pseudouridines. However, the role of its C-terminal tail domain remained unknown. The E265K mutation does not diminish the pseudouridine synthase activity of RluD, or the growth phenotype of Escherichia coli, or cause any detectable defects in the ribosomal assembly in our assays. However, in our in vivo analyses, we observed that the E265K mutation resulted in increased retention of the ribosome binding factor A (RbfA) on 30S suggesting a new role of RluD in contributing to RbfA release, a function which may be attributed to its (RluD) C-terminal tail domain. The studies also reveal that deficiency of RbfA release from 30S compromises the fidelity of i-tRNA selection in the ribosomal P-site.     

Binding of 30S Ribosome Induces Single-stranded Conformation Within and Downstream of the Expression Platform in a Translational Riboswitch

Translational riboswitches are bacterial gene regulatory elements found in the 5′-untranslated region of mRNAs. They operate through a conformational refolding reaction that is triggered by a concentration change of a modulating small molecular ligand. The translation initiation region (TIR) is either released from or incorporated into base pairing interactions through the conformational switch. Hence, initiation of translation is regulated by the accessibility of the Shine-Dalgarno sequence and start codon. Interaction with the 30S ribosome is indispensable for the structural switch between functional OFF and ON states. However, on a molecular level it is still not fully resolved how the ribosome is accommodated near or at the translation initiation region in the context of translational riboswitches. The standby model of translation initiation postulates a binding site where the mRNA enters the ribosome and where it resides until the initiation site becomes unstructured and accessible. We here investigated the adenine-sensing riboswitch from Vibrio vulnificus. By application of a ¹⁹F labelling strategy for NMR spectroscopy that utilizes ligation techniques to synthesize differentially ¹⁹F labelled riboswitch molecules we show that nucleotides directly downstream of the riboswitch domain are first involved in productive interaction with the 30S ribosomal subunit. Upon the concerted action of ligand and the ribosomal protein rS1 the TIR becomes available and subsequently the 30S ribosome can slide towards the TIR. It will be interesting to see whether this is a general feature in translational riboswitches or if riboswitches exist where this region is structured and represent yet another layer of regulation.