Przemysław Grela

Structure and Dynamics of Ribosomal Protein L12: An Ensemble Model Based on SAXS and NMR Relaxation

Ribosomal protein L12 is a two-domain protein that forms dimers mediated by its N-terminal domains. A 20-residue linker separates the N- and C-terminal domains. This linker results in a three-lobe topology with significant flexibility, known to be critical for efficient translation. Here we present an ensemble model of spatial distributions and correlation times for the domain reorientations of L12 that reconciles experimental data from small-angle x-ray scattering and nuclear magnetic resonance. We generated an ensemble of L12 conformations in which the structure of each domain is fixed but the domain orientations are variable. The ensemble reproduces the small-angle x-ray scattering data and the optimized correlation times of its reorientational eigenmodes fit the (15)N relaxation data. The ensemble model reveals intrinsic conformational properties of L12 that help explain its function on the ribosome. The two C-terminal domains sample a large volume and extend further away from the ribosome anchor than expected for a random-chain linker, indicating that the flexible linker has residual order. Furthermore, the distances between each C-terminal domain and the anchor are anticorrelated, indicating that one of them is more retracted on average. We speculate that these properties promote the function of L12 to recruit translation factors and control their activity on the ribosome.

Structural Relationships Among the Ribosomal Stalk Proteins from the Three Domains of Life

The GTPase center of the large ribosomal subunit, being a landing platform for translation factors, and regarded as one of the oldest structures in the ribosome, is a universally conserved structure in all domains of life. It is thought that this structure could be responsible for the major breakthrough on the way to the RNA/protein world, because its appearance would have dramatically increased the rate and accuracy of protein synthesis. The major part of this center is recognized as a distinct structural entity, called the stalk. The main functional part of the stalk in all domains of life is composed of small L12/P proteins, which are believed to form an evolutionarily conserved group. However, some data indicate that the bacterial and archaeo/eukaryal proteins are not related to each other structurally, and only a functional relationship may be clearly recognized. To clarify this point, we performed a comprehensive comparative analysis of the L12/P proteins from the three domains of life. The results show that bacterial and archaeo/eukaryal L12/P-proteins are not structurally related and, therefore, might not be linked evolutionarily either. Consequently, these proteins should be regarded as analogous rather than homologous systems and probably appeared on the ribosomal particle in two independent events in the course of evolution.

Pentameric Organization of the Ribosomal Stalk Accelerates Recruitment of Ricin A Chain to the Ribosome for Depurination

Ribosome inactivating proteins (RIPs) depurinate a universally conserved adenine in the α-sarcin/ricin loop (SRL) and inhibit protein synthesis at the translation elongation step. We previously showed that ribosomal stalk is required for depurination of the SRL by ricin toxin A chain (RTA). The interaction between RTA and ribosomes was characterized by a two-step binding model, where the stalk structure could be considered as an important interacting element. Here, using purified yeast ribosomal stalk complexes assembled in vivo, we show a direct interaction between RTA and the isolated stalk complex. Detailed kinetic analysis of these interactions in real time using surface plasmon resonance (SPR) indicated that there is only one type of interaction between RTA and the ribosomal stalk, which represents one of the two binding steps of the interaction with ribosomes. Interactions of RTA with the isolated stalk were relatively insensitive to salt, indicating that nonelectrostatic interactions were dominant. We compared the interaction of RTA with the full pentameric stalk complex containing two pairs of P1/P2 proteins with its interaction with the trimeric stalk complexes containing only one pair of P1/P2 and found that the rate of association of RTA with the pentamer was higher than with either trimer. These results demonstrate that the stalk is the main landing platform for RTA on the ribosome and that pentameric organization of the stalk accelerates recruitment of RTA to the ribosome for depurination. Our results suggest that multiple copies of the stalk proteins might also increase the scavenging ability of the ribosome for the translational GTPases.

Biophysical Properties of the Eukaryotic Ribosomal Stalk

The landing platform for the translational GTPases is located on the 60S ribosomal subunit and is referred to as a GTPase-associated center. The most distinctive feature of this center is an oligomeric complex, the stalk, responsible for the recruitment of translation factors and stimulation of translation factor-dependent GTP hydrolysis. In eukaryotes, the stalk has been investigated in vitro and in vivo, but most information available concerns its individual components only. In the present study, we provide an insight into the biophysical nature of the native stalk isolated from the yeast Saccharomyces cerevisiae. Using fluorescence, circular dichroism, and mass spectrometry analyses, we were able to characterize the natively formed yeast stalk, casting new light on the oligomeric properties of the complex and its quaternary topology, showing that folding and assembly are coupled processes. The pentameric stalk is an exceptionally stable structure with the protein core composed of P0, P1A, and P2B proteins and less tightly bound P1B and P2A capable of dissociating from the stalk core. We obtained also the whole picture of the posttranslational modifications at the logarithmic phase of yeast growth, using mass spectrometry approach, where P proteins are phosphorylated at a single serine residue, P0 may accept two phosphate groups, and P1A none. Additionally, only P1B undergoes N-terminal acetylation after prior methionine removal.

The P1/P2 proteins of the human ribosomal stalk are required for ribosome binding and depurination by ricin in human cells.

Ricin A‐chain (RTA) depurinates the sarcin–ricin loop of 28S ribosomal RNA and inhibits protein synthesis in mammalian cells. In yeast, the ribosomal stalk facilitates the interaction of RTA with the ribosome and subsequent depurination. Despite homology between the stalk structures from yeast and humans, there are notable differences. The human ribosomal stalk contains two identical heterodimers of P1 and P2 bound to P0, whereas the yeast stalk consists of two different heterodimers, P1α–P2β and P2α–P1β, bound to P0. RTA exhibits higher activity towards mammalian ribosomes than towards ribosomes from other organisms, suggesting that the mode of interaction with ribosomes may vary. Here, we examined whether the human ribosomal stalk proteins facilitate the interaction of RTA with human ribosomes and subsequent depurination of the sarcin–ricin loop. Using small interfering RNA‐mediated knockdown of P1/P2 expression in human cells, we demonstrated that the depurination activity of RTA is lower when P1 and P2 levels are reduced. Biacore analysis showed that ribosomes from P1/P2‐depleted cells have a reduced ability to bind RTA, which correlates with reduced depurination activity both in vitro and inside cells. RTA interacts directly with recombinant human P1–P2 dimer, further demonstrating the importance of human P1 and P2 in enabling RTA to bind and depurinate human ribosomes.

Sexing Red-Necked Grebes Podiceps grisegena by Molecular Techniques and Morphology

Abstract. Sexual size dimorphism was analysed in the Red- necked Grebe in southeast Poland. A DNA-based procedure was utilised to sex individuals and to assess the accuracy of morphological criteria for the sex identification of adult breeding birds: discriminant analysis on the sample level and within-pair comparisons. Males were significantly larger than females in all body measurements used in the discriminant function selection process. Owing to considerable overlap in measurements, however, the sexes cannot be accurately separated by biometrics at the population scale. Sexual dimorphism was most pronounced in bill length measured from the corner of the gape to the tip, but only 79% of individuals were correctly identified on the basis of this parameter alone. When two variables, bill length and wing length, were combined, the discriminant function was of similar efficiency (80%) in determining the sex. The accuracy level of sexing may be improved by comparing mates within pairs: combined comparisons of bill length and body mass were as accurate as the genetic technique, but sex assignment was restricted to 76% of the measured pairs.

Subcellular localization of ribosomal P0-like protein MRT4 is determined by its N-terminal domain.

The Mrt4 protein, showing extensive sequence similarity to the ribosomal P0 protein, is classified as a ribosomal P0-like protein and acts as a trans-acting factor which modulates the assembly of the pre-60S particle. In this report we investigated the biological nature of the human Mrt4 protein. First, we constructed a series of hybrid hMrt4-P0 proteins by replacing various domains of the P0 protein with corresponding protein fragments from hMrt4. We found that hMrt4 binds to the same site on the large ribosomal subunit as does P0, but despite the sequence homology it is not able to functionally complement the lack of P0. Using fluorescence microscopy and biochemical approaches we also show that hMrt4 occupies predominantly the nucleolar compartment, in contrast to P0 and P1/P2, which are located in the cytoplasm. The nucleolar accumulation of hMrt4 does not depend on a specific nucleolus localization signal, but rather occurs via interaction with established nucleolar components such as rRNA; however, nuclear import of hMrt4 is dependent on a short sequence in the N-terminal part of the protein. Functional analysis with specific inhibitors, actinomycin D and leptomycin B, showed that hMrt4 is a trans-acting factor involved in ribosome maturation, with nucleus-cytoplasm shuttling capacity.

The role of male nest building in post-mating sexual selection in the monogamous red-necked grebe

In monogamous birds, early male parental effort, such as nest building, may serve as a post-mating sexually-selected display allowing female assessment of male quality. We examined the functional significance of male nest building and the potential role of nest size as a sexually-selected signal in the red-necked grebe (Podiceps grisegena), a species with high mate fidelity. Time-activity budgets showed that no behaviour was performed exclusively by one sex in the pre-laying period, but males spent significantly more time nest building and were more often involved in aggressive intra- and interspecific interactions. Nest building in pairs attempting a second brood was also performed predominantly by males. Greater participation in nest construction by males allowed females to allocate more time to self-maintenance activities in the period prior to egg-laying. The positive relationship found between the relative contribution of males to nest building and later to brood provisioning indicates that male nest building is an honest indicator of future paternal effort. Males obtained copulations solicited by females proportionally to the time spent on nest building, and the extent of male participation in nest construction was of importance for explaining variation in clutch size. Nest size itself is not very likely to be sexually selected in red-necked grebes, as it was found to depend on nest site conditions such as water depth and exposure to wave action. We suggest that greater investment of males in energetically demanding pre-laying activities is functionally similar to post-mating courtship feeding; it constitutes males’ indirect contribution to clutch production and may help to negotiate the relative investment each sex makes in the different stages of the breeding cycle. The results support the idea that, in monogamous birds, naturally selected male characters related to parental care may evolve into important sexual signals to females, although not into extreme displays.

Solution structure of the natively assembled yeast ribosomal stalk determined by small-angle X-ray scattering

The ribosomal stalk of the 60S subunit has been shown to play a crucial role in all steps of protein synthesis, but its structure and exact molecular function remain an unanswered question. In the present study, we show the low-resolution models of the solution structure of the yeast ribosomal stalk, composed of five proteins, P0-(P1-P2)(2). The model of the pentameric stalk complex determined by small-angle X-ray scattering reveals an elongated shape with a maximum length of 13 nm. The model displays three distinct lobes, which may correspond to the individual P1-P2 heterodimers anchored to the C-terminal domain of the P0 protein.

Hematological indicators of habitat quality: Erythrocyte parameters reflect greater parental effort of Red-necked Grebes under ecological trap conditions

ABSTRACT Knowledge about interactions between habitat quality and individual condition is vital for understanding ecological processes. We examined variation in commonly used hematological indices and body mass in incubating and chick-rearing Red-necked Grebes (Podiceps grisegena) in Poland breeding under contrasting foraging conditions in ponds used for aquaculture. Grebes used either ponds with low prey availability in the early breeding period and abundant food in the brood-rearing stage (high-quality habitat), or ponds stocked with fish attractive as prey to spring-arriving adults yet later unavailable to chicks due to size constraints (low-quality habitat acting as an ecological trap). Parental effort, measured as the proportion of time allocated to food acquisition and provisioning of chicks, was greater in the low-quality habitat, although adult grebes spent less time foraging there than in the high-quality habitat early in the season. Scaled body mass was higher in incubating grebes than in brood-rearing grebes, regardless of habitat quality. Breeding stage by habitat interactions were significant for hemoglobin, erythrocyte count (RBC), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH), with greater MCV and MCH values (by 20–34%, on average) during brood-rearing than during incubation in the low-quality habitat, and the opposite pattern (by 15–16%) in the high-quality habitat. These hematological responses signify changes in physiological stress associated with parental workloads. Higher MCV and MCH levels (by 22–31%) during the chick-rearing stage in the low-quality compared with the high-quality habitat indicate that parents incurred physiological costs of breeding under ecological trap conditions. No significant effects were found on leucocyte indices (total leucocyte count and heterophil/lymphocyte ratio). We consider the hemoglobin- and RBC-related parameters to be promising indicators of habitat-driven stress associated with prolonged physical exertion, especially when multiple metrics are employed.