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991.
Viral replication initiator proteins are multifunctional proteins that utilize ATP binding and hydrolysis by their AAA+ modules for multiple functions in the replication of their viral genomes. These proteins are therefore of particular interest for understanding how AAA+ proteins carry out multiple ATP driven functions. We have performed a comprehensive mutational analysis of the residues involved in ATP binding and hydrolysis in the papillomavirus E1 initiator protein based on the recent structural data. Ten of the eleven residues that were targeted were defective for ATP hydrolysis, and seven of these were also defective for ATP binding. The three mutants that could still bind nucleotide represent the Walker B motif (D478 and D479) and Sensor 1 (N523), three residues that are in close proximity to each other and generally are considered to be involved in ATP hydrolysis. Surprisingly, however, two of these mutants, D478A and N523A, mimicked the nucleotide bound state and were capable of binding DNA in the absence of nucleotide. However, these mutants could not form the E1 double trimer in the absence of nucleotide, demonstrating that there are two qualitatively different consequences of ATP binding by E1, one that can be mimicked by D478A and N523A and one which cannot.Viral initiator proteins from DNA viruses belong to the superfamily 3 (SF3) helicases (5, 9). Well-studied members of this group include the T-antigens from the polyomaviruses, the E1 proteins from the papillomaviruses, and the Rep proteins from the adeno-associated viruses. These proteins are multifunctional proteins that utilize ATP binding and hydrolysis by their AAA+ (ATPases associated with various cellular activities) modules for multiple functions in the replication of their viral genomes. AAA+ modules are ∼250-amino-acid ATP binding domains that carry out numerous ATP driven functions (for reviews, see references 6 and 7). For example, the E1 protein, which plays an essential role in papillomavirus DNA replication, has multiple functions that are affected by binding or hydrolysis of ATP (14, 18, 21, 23, 24, 26). E1 is a DNA-binding protein, which binds specifically to E1 binding sites (E1 BS) in the origin of DNA replication (2, 8, 15, 22, 25). DNA-binding activity requires nucleotide binding by E1 (15). In the presence of ATP or ADP, E1 can form a specific double-trimer (DT) complex on the ori and, through ATP hydrolysis, this complex can melt the ori DNA (13, 15, 16). In a process that requires ATP hydrolysis, the DT is then converted into a double hexamer (DH), which has ATP-dependent DNA helicase activity and is the replicative DNA helicase (15, 26). Consequently, the E1 AAA+ module is utilized for ATP binding and hydrolysis in at least two different E1 complexes with different functions. An interesting question is how the same motif for ATP binding and hydrolysis is used in these different complexes to achieve their differing functions.Structural studies of representatives from all three groups—E1 proteins, T antigens, and Rep proteins—have provided important information about how ATP is bound and hydrolyzed by these proteins and the structural consequences that result (1, 4, 7, 10-12). For example, in the recent crystal structure of a hexamer of the E1 oligomerization and helicase domains formed on single-stranded DNA, an ATP binding pocket is formed by 11 residues from two adjacent monomers of the E1 helicase domain (Fig. (Fig.1A)1A) (3). Because most of the residues thought to be involved in ATP binding and hydrolysis in these AAA+ proteins are highly conserved and form particular substructures, the specific function of the individual residues have been predicted for these proteins (6) (see Fig. Fig.1A).1A). It is well established that the conserved residues in the Walker A and Walker B motifs are involved in both binding and hydrolysis of ATP. The Sensor 1 residues are generally involved in contacting Walker B and the γ-phosphate of ATP. The Sensor 2 motif also participates in nucleotide binding and interacts directly with the γ-phosphate of ATP.Open in a separate windowFIG. 1.(A) Residues in E1 involved in nucleotide binding and hydrolysis. A schematic image is shown of the interface between two E1 monomers that constitute the ATP binding pocket of BPV E1 with the residues that are predicted to be involved in ATP binding and hydrolysis (adapted from reference 3). The Walker A, Walker B, and Sensor 1 and Sensor 2 motifs and the arginine finger are indicated. (B) Formation of the E12E22-ori complex. EMSA was performed using an 84-bp ori probe. Two quantities (1.5 and 3 ng) of wt E1 and of each E1 substitution, as indicated at the top of the gels, were used in the presence of 0.1 ng of full-length E2. In lane 23, E2 alone was added. The mobility of the E22 and E12E22 complexes are indicated. (C) ATPase activity of E1 substitution mutations of residues involved in ATP binding and hydrolysis. Portions (80 ng) of wt E1 or of each respective E1 substitution mutant as indicated were tested for ATPase activity using 32P-labeled γ-ATP. After the reaction the free phosphate was separated from ATP by thin-layer chromatography and quantitated by using a Fuji imager. Lane 12 contained [γ-32P]ATP only.To gain a more precise understanding of the role of these particular residues in ATP binding and hydrolysis and because a systematic analysis of such residues has not been performed for the E1 initiator proteins, we performed a mutational analysis of these 11 residues. Based on the behavior of mutants in these residues, the residues can be classified into three groups. Seven of the mutations result in a protein that fails to bind nucleotide and consequently also fail to hydrolyze nucleotide. Three mutants can still bind nucleotide but fail to hydrolyze ATP. Surprisingly, two of these mutants mimic the ATP-bound state and can bind DNA in the absence of nucleotide, demonstrating that E1 utilizes ATP binding for two different modes, only one of which can be mimicked by the mutations in the ATP binding pocket. 相似文献
992.
A new method for CO2 supply to photoautotrophic organisms was developed, and its applicability for measuring specific growth rates in shaken batch cultures of cyanobacteria and unicellular algae was shown. Small bags containing a concentrated carbonate buffer with a CO2 partial pressure of 32 mbar were prepared from a thin foil of low density polyethylene (LDPE). These bags were inserted as CO2 reservoirs (CRs) into polystyrene culture flasks with gas‐permeable screw caps, which were suitable to photometric growth measurement. CO2 was released directly into the medium with membrane‐controlled kinetics. The CRs were not depleted within 1 week, although the atmosphere in the culture vessel exchanged rapidly with the ambient air. Rates of initial growth and final densities of the cultures of six different unicellular algal species and one cyanobacterium were markedly increased by diffusive CO2 supply from the CR. In the presence of a CR, growth was exponential during the first 2 d in all cultures studied. The method described allowed a high number of measurements of specific growth rates with relatively simple experimental setup. 相似文献
993.
The significant role of space in maintaining species coexistence and determining community structure and function is well established. However, community ecology studies have mainly focused on simple competition and predation systems, and the relative impact of positive interspecific interactions in shaping communities in a spatial context is not well understood. Here we employ a spatially explicit metacommunity model to investigate the effect of local dispersal on the structure and function of communities in which species are linked through an interaction web comprising mutualism, competition and exploitation. Our results show that function, diversity and interspecific interactions of locally linked communities undergo a phase transition with changes in the rate of species dispersal. We find that low spatial interconnectedness favors the spontaneous emergence of strongly mutualistic communities which are more stable but less productive and diverse. On the other hand, high spatial interconnectedness promotes local biodiversity at the expense of local stability and supports communities with a wide range of interspecific interactions. We argue that investigations of the relationship between spatial processes and the self-organization of complex interaction webs are critical to understanding the geographic structure of interactions in real landscapes. 相似文献
994.
995.
Geertje J. Janssen Eugenio Daviso Martin van Son Huub J. M. de Groot A. Alia Jörg Matysik 《Photosynthesis research》2010,104(2-3):275-282
Cyanobacteria are widely used as model organism of oxygenic photosynthesis due to being the simplest photosynthetic organisms containing both photosystem I and II (PSI and PSII). Photochemically induced dynamic nuclear polarization (photo-CIDNP) 13C magic-angle spinning (MAS) NMR is a powerful tool in understanding the photosynthesis machinery down to atomic level. Combined with selective isotope enrichment this technique has now opened the door to study primary charge separation in whole living cells. Here, we present the first photo-CIDNP observed in whole cells of the cyanobacterium Synechocystis. 相似文献
996.
Lene Songe-M?ller Erwin van den Born Vibeke Leihne Cathrine B. V?gb? Terese Kristoffersen Hans E. Krokan Finn Kirpekar P?l ?. Falnes Arne Klungland 《Molecular and cellular biology》2010,30(7):1814-1827
Uridines in the wobble position of tRNA are almost invariably modified. Modifications can increase the efficiency of codon reading, but they also prevent mistranslation by limiting wobbling. In mammals, several tRNAs have 5-methoxycarbonylmethyluridine (mcm5U) or derivatives thereof in the wobble position. Through analysis of tRNA from Alkbh8−/− mice, we show here that ALKBH8 is a tRNA methyltransferase required for the final step in the biogenesis of mcm5U. We also demonstrate that the interaction of ALKBH8 with a small accessory protein, TRM112, is required to form a functional tRNA methyltransferase. Furthermore, prior ALKBH8-mediated methylation is a prerequisite for the thiolation and 2′-O-ribose methylation that form 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) and 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm5Um), respectively. Despite the complete loss of all of these uridine modifications, Alkbh8−/− mice appear normal. However, the selenocysteine-specific tRNA (tRNASec) is aberrantly modified in the Alkbh8−/− mice, and for the selenoprotein Gpx1, we indeed observed reduced recoding of the UGA stop codon to selenocysteine.tRNAs are frequently modified at the wobble uridine, a feature that is believed to either promote or restrict wobbling depending on the type of modification. In the case of eukaryotes, the functions of wobble uridine modifications have been studied in the greatest detail in Saccharomyces cerevisiae. Here, the modifications 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), and 5-carbamoylmethyluridine (ncm5U) or its 2′-O-ribose-methylated form, ncm5Um, are found in 11 out of 13 wobble uridine-containing tRNAs (22). mcm5U and mcm5s2U are mostly found in “split” codon boxes, where the pyrimidine- and purine-ending codons encode different amino acids, while ncm5U is found in “family” codon boxes, where all four codons encode a single amino acid. Early reports based on in vitro experiments suggested that wobble nucleosides, such as mcm5U, ncm5U, and their derivatives, may restrict wobbling (17, 37, 45), but the results of a recent comprehensive study performed in vivo in S. cerevisiae show that such modifications can improve the reading both of the cognate, A-ending codons and of the wobble, G-ending codons (22). This may suggest that the primary role of these modified nucleosides is to improve translational efficiency rather than to restrict wobbling.The characterization of wobble uridine modifications in higher eukaryotes is very limited, and little is known about the enzymes that introduce them. In mammals, mcm5s2U has been found in the wobble position of tRNAGlu(UUC), tRNALys(UUU), and tRNAArg(UCU) (40). Unlike yeast, mammals possess a specialized tRNA that is responsible for recoding the UGA stop codon to insert the 21st amino acid, selenocysteine (Sec). The mammalian tRNASec population consists of two subpopulations containing either mcm5U or the ribose-methylated derivative mcm5Um in the wobble position. Interestingly, ribose methylation of mcm5U in tRNASec appears to have a role in regulating selenoprotein synthesis, as the expression of some selenoproteins, such as glutathione peroxidase 1 (Gpx1), appears to be promoted by mcm5Um-containing tRNASec (5, 7, 9, 32).Some years ago, the Escherichia coli AlkB protein was found to be a 2-oxoglutarate- and iron-dependent dioxygenase capable of demethylating the lesions 1-methyladenosine and 3-methylcytosine in DNA (13, 42). Multicellular organisms generally possess several different AlkB homologues (ALKBH), and bioinformatics analysis has identified eight different mammalian ALKBH proteins, denoted ALKBH1 to ALKBH8 in humans and Alkbh1 to Alkbh8 in mice, as well as the somewhat-less-related, obesity-associated FTO protein (2, 16, 30). Among the ALKBH proteins of unknown function, ALKBH8 is the only one containing additional annotated protein domains. Here, the AlkB domain is localized between an N-terminal RNA recognition motif (RRM) and a C-terminal methyltransferase (MT) domain. Interestingly, the MT domain has sequence homology to the S. cerevisiae tRNA methyltransferase Trm9, which has been shown to catalyze the methyl esterification of modified wobble uridine (U34) residues of tRNAArg and tRNAGlu, resulting in the formation of mcm5U and mcm5s2U, respectively (23, 43). Until recently, human ALKBH8 was incorrectly annotated in the protein sequence database, and another human protein, KIAA1456, has been designated the human Trm9 homologue (3, 23).We have generated for this study Alkbh8-targeted mice that lack exons critical for both the MT and AlkB activities of Alkbh8. The mice did not display any overt phenotype, but tRNA from these mice was completely devoid of mcm5U, mcm5s2U, and mcm5Um, and the relevant tRNA isoacceptors instead contained the acid form 5-carboxymethyluridine (cm5U) and/or the amide forms ncm5U/ncm5s2U. Furthermore, we show that recombinant ALKBH8 and TRM112 form a heterodimeric complex capable of catalyzing the methyl esterification of cm5U and cm5s2U to mcm5U and mcm5s2U, respectively. In agreement with the involvement of mcm5Um in selenoprotein synthesis, we observed a reduced level of Gpx1 in the Alkbh8−/− mice, and tRNASec from these mice showed a reduced ability to decode the UGA stop codon to Sec. 相似文献
997.
Johan Reinert Vikan Bård Gunnar Stokke Jarkko Rutila Esa Huhta Arne Moksnes Eivin Røskaft 《Evolutionary ecology》2010,24(5):1141-1157
The brood parasitic common cuckoo Cuculus canorus has a history of coevolution that involves numerous passerine hosts, but today only a subset is known to be regularly parasitised
in any area. In some hosts, there is significant variation in the occurrence of parasitism between populations, but still
individuals in non-parasitised populations show strong antiparasite defences. In the present study we compared the strength
of egg rejection of four distant Fennoscandian brambling Fringilla montifringilla populations experiencing different levels of cuckoo parasitism (0–6%). Egg rejection ability was in general very well developed
and we did not find any population differences in the relationship between egg rejection probability and similarity between
host and experimental parasitic eggs. Furthermore, bramblings very rarely made errors in rejection, indicating that selection
against rejection behaviour is likely to be very weak. The brambling-cuckoo system therefore differs from other well studied
systems which are characterised by pronounced spatial and temporal variation in the host’s level of defence. This result is
unlikely to reflect independent replication of the same evolutionary trajectory because the weak breeding site tenacity of
bramblings should result in an extreme amount of gene flow within the distribution area and thus strongly impede localised
responses to selection. Instead, lack of geographic variation has more likely arisen because bramblings respond to selection
as one evolutionary unit, and because the average parasitism pressures have been high enough in the past to cause regional
fixation of rejection alleles and evolution of clutch characteristics that facilitate cost free egg recognition. 相似文献
998.
Roberta E Redfern Marie-Claire Daou Li Li Mary Munson Arne Gericke Alonzo H Ross 《Protein science : a publication of the Protein Society》2010,19(10):1948-1956
The tumor suppressor, phosphatase, and tensin homologue deleted on chromosome 10 (PTEN), is a phosphoinositide (PI) phosphatase specific for the 3‐position of the inositol ring. PTEN has been implicated in autism for a subset of patients with macrocephaly. Various studies identified patients in this subclass with one normal and one mutated PTEN gene. We characterize the binding, structural properties, activity, and subcellular localization of one of these autism‐related mutants, H93R PTEN. Even though this mutation is located at the phosphatase active site, we find that it affects the functions of neighboring domains. H93R PTEN binding to phosphatidylserine‐bearing model membranes is 5.6‐fold enhanced in comparison to wild‐type PTEN. In contrast, we find that binding to phosphatidylinositol‐4,5‐bisphosphate (PI(4,5)P2) model membranes is 2.5‐fold decreased for the mutant PTEN in comparison to wild‐type PTEN. The structural change previously found for wild‐type PTEN upon interaction with PI(4,5)P2, is absent for H93R PTEN. Consistent with the increased binding to phosphatidylserine, we find enhanced plasma membrane association of PTEN‐GFP in U87MG cells. However, this enhanced plasma membrane association does not translate into increased PI(3,4,5)P3 turnover, since in vivo studies show a reduced activity of the H93R PTEN‐GFP mutant. Because the interaction of PI(4,5)P2 with PTEN's N‐terminal domain is diminished by this mutation, we hypothesize that the interaction of PTEN's N‐terminal domain with the phosphatase domain is impacted by the H93R mutation, preventing PI(4,5)P2 from inducing the conformational change that activates phosphatase activity. 相似文献
999.
1000.
Katherina L. Schoo Nicole Aberle Arne M. Malzahn Maarten Boersma 《Aquatic Ecology》2010,44(1):233-242
We investigated whether phosphorus limitations of primary producers propagate upwards through the food web, not only to the
primary consumer level but also onto the secondary consumers’ level. A tri-trophic food chain was used to assess the effects
of phosphorus-limited phytoplankton (the cryptophyte Rhodomonas salina) on herbivorous zooplankters (the copepod Acartia tonsa) and finally on zooplanktivores (the ctenophore Pleurobrachia pileus). The algae were cultured in phosphorus-replete and phosphorus-limited media before being fed to two groups of copepods.
The copepods in turn were fed to the top predator, P. pileus, in a mixture resulting in a phosphorus-gradient, ranging from copepods having received only phosphorus-replete algae to
copepods reared solely on phosphorus-limited algae. The C:P ratio of the algae varied significantly between the two treatments,
resulting in higher C:P ratios for those copepods feeding on phosphorus-limited algae, albeit with a significance of 0.07.
The differences in the feeding environment of the copepods could be followed to Pleurobrachia pileus. Contrary to our expectations, we found that phosphorus-limited copepods represented a higher quality food source for P. pileus, as shown by the better condition (expressed as nucleic acid content) of the ctenophore. This could possibly be explained
by the rather high C:P ratios of ctenophores, their resulting low phosphorus demand and relative insensitivity to P deficiency.
This might potentially be an additional explanation for the observed increasing abundances of gelatinous zooplankton in our
increasingly phosphorus-limited coastal seas. 相似文献