Protein synthesis in Archaea. mechanism of dissociation from ribosomes of the conserved translation factor aIF6; development of an in vitro transcription/translation system from the thermophilic archaeon Sulfolobus solfataricus

This work describes two experimental studies performed for my doctoral thesis. The general subject is the analysis of the features and mechanisms of protein synthesis in extremely thermophilic Archaea. The subject is interesting and relevant from different points of view. Firstly, it has long been known that archaeal translation shares several features and components with the corresponding eukaryal process. Investigating the common themes between archaeal and eukaryal protein synthesis may help to shed light on the evolutionary origin thereof and to reconstruct the pathways whereby Eukarya emerged from the common tree of life. Secondly, extremely thermophilic Archaea have a lot of potential for biotechnological development, for instance as a source of thermostable enzymes to be used for both medical and industrial purposes. In the first part of my work, I shall describe a functional analysis of the archaeal translation factor aIF6, a protein shared by the Archaea and the Eukarya but not found in Bacteria. Although the eukaryal factor (eIF6) has been studied extensively, its function in translation is still imperfectly understood. It is established that it acts as a ribosome anti-association factor, binding to the large subunit and preventing its premature joining with the small subunit. To allow the 60S ribosome to enter the translation cycle, eIF6 must be actively released, a process that depends on the action of another factor, Sdo1/SBDS, which is also found in Archaea, and of a GTPase, EFL1, that instead does not have an archaeal homologue. In my work, I studied the mechanism of aIF6 release from archaeal large ribosomal subunits, using as the model organism the thermophilic archaeon Sulfolobus solfataricus. The results I obtained show that detachment of aIF6 from ribosomes requires the GTPase activity of elongation factor 2 (aEF2), while the Sdo1/SBDS homologue apparently inhibits rather than promoting aIF6 release. The function of archaeal Sdo1 remains therefore to be understood and must be studied further. In the second part of work, I developed a coupled in vitro transcription/translation system for cell-free protein synthesis from cell lysates of S. solfataricus. The essential element in this expression system is a strong promoter derivedfrom 16S/23S rRNA-encoding DNA promoter from the archaebacterium Sulfolobus sp. P2 that produces, with high efficiency, specific mRNAs. I show that this method permits the efficient synthesis in vitro at high temperature of biologically active proteins

Optimization of an in vitro transcription/translation system based on Sulfolobus solfataricus cell lysate.

A system is described which permits the efficient synthesis of proteins in vitro at high temperature. It is based on the use of an unfractionated cell lysate (S30) from Sulfolobus solfataricus previously well characterized in our laboratory for translation of pre-transcribed mRNAs, and now adapted to perform coupled transcription and translation. The essential element in this expression system is a strong promoter derived from the S. solfataricus 16S/23S rRNA-encoding gene, from which specific mRNAs may be transcribed with high efficiency. The synthesis of two different proteins is reported, including the S. solfataricus DNA-alkylguanine-DNA-alkyl-transferase protein (SsOGT), which is shown to be successfully labeled with appropriate fluorescent substrates and visualized in cell extracts. The simplicity of the experimental procedure and specific activity of the proteins offer a number of possibilities for the study of structure-function relationships of proteins

The archaeal elongation factor EF-2 induces the release of aIF6 from 50S ribosomal subunit

The translation factor IF6 is a protein of about 25 kDa shared by the Archaea and the Eukarya but absent in Bacteria. It acts as a ribosome anti-association factor that binds to the large subunit preventing the joining to the small subunit. It must be released from the large ribosomal subunit to permit its entry to the translation cycle. In Eukarya, this process occurs by the coordinated action of the GTPase Efl1 and the docking protein SBDS. Archaea do not possess a homolog of the former factor while they have a homolog of SBDS. In the past, we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homolog (aIF6) highlighting its similarity to the eukaryotic counterpart. Here, we analyzed the mechanism of aIF6 release from the large ribosomal subunit. We found that, similarly to the Eukarya, the detachment of aIF6 from the 50S subunit requires a GTPase activity which involves the archaeal elongation factor 2 (aEF-2). However, the release of aIF6 from the 50S subunits does not require the archaeal homolog of SBDS, being on the contrary inhibited by its presence. Molecular modeling, using published structural data of closely related homologous proteins, elucidated the mechanistic interplay between the aIF6, aSBDS, and aEF2 on the ribosome surface. The results suggest that a conformational rearrangement of aEF2, upon GTP hydrolysis, promotes aIF6 ejection. On the other hand, aSBDS and aEF2 share the same binding site, whose occupation by SBDS prevents aEF2 binding, thereby inhibiting aIF6 release

Development and testing of a model for risk and protective factors for eating disorders and higher weight among emerging adults: A study protocol

© 2019 Research has demonstrated that eating disorders (ED) and higher weight have lifetime co-occurrence suggesting that they may be best considered within a common etiological model. Although we know that body dissatisfaction is likely to be a risk factor for both outcomes, other proposed risk and protective factors for each condition have not been adequately explored. The current paper tests a conceptual model that is based on a review of the existing literature from both areas of scholarship. It considers biological, sociocultural, psychological, and behavioral factors that may contribute to both outcomes. The model will be tested in a longitudinal design with an initial sample of 600 emerging adults (aged 18–30) per country in nine different countries (total sample = 5400 participants). Questionnaires will be completed online on two occasions, 12 months apart. The first full phase of the study commenced in July 2018, the same time Body Image was approached to publish this protocol paper (the final revised paper was submitted in September 2019), and data collection will be finalized in December 2019. Multi-group path analysis will identify the biopsychosocial predictors – both cross-sectionally and longitudinally – of both ED and higher weight, and how these vary across countries and gender

Optimization of an In Vitro Transcription/Translation System Based on Sulfolobus solfataricus Cell Lysate

A system is described which permits the efficient synthesis of proteins in vitro at high temperature. It is based on the use of an unfractionated cell lysate (S30) from Sulfolobus solfataricus previously well characterized in our laboratory for translation of pretranscribed mRNAs, and now adapted to perform coupled transcription and translation. The essential element in this expression system is a strong promoter derived from the S. solfataricus 16S/23S rRNA-encoding gene, from which specific mRNAs may be transcribed with high efficiency. The synthesis of two different proteins is reported, including the S. solfataricus DNA-alkylguanine-DNA-alkyl-transferase protein (SsOGT), which is shown to be successfully labeled with appropriate fluorescent substrates and visualized in cell extracts. The simplicity of the experimental procedure and specific activity of the proteins offer a number of possibilities for the study of structure-function relationships of proteins

Psychometric properties of measures of sociocultural influence and internalization of appearance ideals across eight countries.

The aim of the current study was to examine the psychometric properties of two well-established measures of sociocultural influence and internalization of the thin/low body fat ideal and muscular ideal. Data from 6272 emerging adults (68.9 % female), aged 18-30 years from Australia, Belgium, Canada, China, Italy, Japan, Spain, and the U.S. were included in this study. Participants completed measures of pressure from mother, fathers, peers, and media, to both increase muscles and lose weight, as well as internalization of the thin/low body fat ideal and muscular ideal. Overall, support for partial invariance was found across the scales. In addition, group level differences were found between countries as well as along demographic factors including gender, age, body mass index, and socioeconomic status. These findings make an important contribution by identifying these scales as useful tools that will support future cross-country and cross-cultural examinations of explanatory models of the development of body image and eating concerns grounded within sociocultural theories.status: Published onlin

Psychometric properties of measures of sociocultural influence and internalization of appearance ideals across eight countries

The aim of the current study was to examine the psychometric properties of two well-established measures of sociocultural influence and internalization of the thin/low body fat ideal and muscular ideal.Data from 6272 emerging adults (68.9 % female), aged 18–30 years from Australia, Belgium, Canada,China, Italy, Japan, Spain, and the U.S. were included in this study. Participants completed measures of pressure from mother, fathers, peers, and media, to both increase muscles and lose weight, as well as internalization of the thin/low body fat ideal and muscular ideal. Overall, support for partial invariance was found across the scales. In addition, group level differences were found between countries as well as along demographic factors including gender, age, body mass index, and socioeconomic status. These findings make an important contribution by identifying these scales as useful tools that will support future cross-country and cross-cultural examinations of explanatory models of the development of bodyimage and eating concerns grounded within sociocultural theories

Cross-Country Measurement Invariance and Effects of Sociodemographic Factors on Body Weight and Shape Concern-Related Constructs in Eight Countries

The aim of this study was to examine the cross-country invariance of five well-established measures of body weight and shape concern-related attitudes and behaviors (i.e., drive for leanness, drive for muscularity, strategies to increase muscle, strategies to lose weight, and weight and shape concerns). A secondary objective was to examine the effects of several sociodemographic factors (age, BMI, socioeconomic status, and gender) on item and latent factor scores of these constructs. A total of 6272 emerging adults (4218 women; Mage = 21.46, SD = 3.11) from Australia, Belgium, Canada, China, Italy, Japan, Spain, and the U.S. completed a self-report online survey as part of a larger study. Overall, support for partial invariance both across countries and in terms of the considered sociodemographic factors was found for reduced versions of the five measures. Significant differences in latent means were found across countries, these being of greater magnitude for drive for leanness and strategies to lose weight. The considered sociodemographic factors (most notably BMI and gender) were associated with the latent mean scores of the assessed constructs. The present study contributes to current literature by providing cross-cultural invariant versions of several measures of relevance in the field of body image.status: publishe

Measuring perfectionism,impulsivity, self-esteem and social anxiety: cross-national study in emerging adults from eight countries.

This study assessed the measurement invariance of selected self-report measures of perfectionism,impulsivity, self-esteem and social anxiety in samples of emerging adults from eight different countries. Participants (N = 6272) completed the Multidimensional Perfectionism Scale (MPS), the UPPS-P Impulsive Behavior Scale, the 5-item Rosenberg Self-Esteem Scale (5-item RSES) and the Social Interaction Anxiety Scale (SIAS-6). Multigroup confirmatory factor analysis was run to examine cross-measure equivalence. Evidence for structural invariance was found for all questionnaires, while weak invariance was supported only for the 5-item RSES and the SIAS-6, and a partial weak invariance was found for the MPS and the UPPS-P. No measure achieved strong invariance. Strict invariance was achieved for the5-item RSES, whereas only a partial strict invariance was supported for the MPS, UPPS-P and SIAS-6.These results suggest that perfectionism, impulsivity, self-esteem, and social anxiety are subjected to linguistic/cultural influence as well as to the effects of socio-demographic variables and can be evaluated by using the selected measures in eight different countries worldwide - but results should be interpreted with caution