Atom transfer radical polymerization (ATRP) is a controlled/living polymerization process used to synthesize polymers with controlled molecular weight and narrow polymer distributions. Control of these key parameters allows for the fabrication of well-defined macromolecular structures, a necessary tool for the synthesis of advanced materials. Since its discovery in 1995, ATRP has received considerable interest and widespread adoption from the academic community. Unfortunately, it faces several complex challenges which have hindered its full scale commercialization, mainly its high catalyst loadings to obtain fast reaction kinetics. One of the premises of this research project was to augment the slow reaction rates of ATRP while using extremely low catalyst concentrations. A hybrid ATRP system was employed which encompassed the fast reaction kinetics associated with conventional free radical processes, with the attractive control features of ATRP. When high free radical initiator concentrations in the range of 0.1 M to 0.2 M were used in concert with ATRP, fast reaction rates were realized, while maintaining a polymerization with living characteristics. Conversions of 81% (0.117M) and 91% (0.234M) were achieved within 2 hours as compared to typical ATRPs where achieving such conversions would take up to 24 hours. For those same free radical initiator loadings (0.117M and 0.234M) the reaction demonstrated living characteristics with molecular weight growing in a linear fashion with respect to increasing monomer conversion. Despite the high free radical initiator concentration, the polymer distribution remained relatively narrow, not exceeding a polydispersity of 1.30. Chain extension experiments from a synthesized macroinitiator were successful which demonstrated the living characteristics of the hybrid ATRP process. The aforementioned polymerizations were conducted with various copper concentrations. Catalyst concentrations as low as 16 ppm (0.234mM) were found to be effective, i.e. one catalyst mediated the growth of over 100 polymer chains, and thus saving post polymerization purification. Moreover, the expensive ligand cost could be cut dramatically through a nearly 100 time reduction in the ligand concentration for these polymerizations. A hybrid ATRP system was used as a unique method to determine termination rate coefficients of MMA at 70°C as a function of both conversion and chain length. A three dimensional composite map was developed to elucidate the coupling effects of both conversion and chain length on the termination rate coefficient over a total range of data which can be used for modelling systems of this nature. / Thesis / Master of Applied Science (MASc)

Biennial bearing has been investigated longer and more extensively in apple than in any other fruit tree; however, it remains a serious problem in commercial apple production all over the world. Trees that have become biennial flower profusely and carry a heavy crop in the "on" year, and flower sparsely or not at all and carry little or no crop the following year, the "off" year. Fruit in the "on" year tend to be small, poorly colored, and of low quality, while the few fruit in the "off" year are usually too large, become susceptible to physiological disorders, and also are of poor quality. Without intervention, the crops in both the "on" and "off" years are undesirable and uneconomical. The most common method used by commercial apple growers to try to prevent biennial bearing is chemical fruit thinning, which is an "on" year method of removing a part of the crop before it matures on the tree. In general, growers don't do anything in the "off" year to prevent biennial bearing with the exceptions of fertilizing and pruning lightly. In this study, several experiments were conducted with the cultivars "Braeburn", "Golden Delicious", "Ramey York", and "Fuji" in the "off" year to try and suppress FBI and thus prevent a biennial bearing situation in the following year. The first set of experiments studied the effect of whole-tree and partial-tree defoliation on suppressing spur and lateral flowering and fruit set. Flowering and fruit set were suppressed with defoliation in most cases. Defoliation in early July caused the least amount of flowering the following year and in some cases it was zero. As the defoliation timing and severity was delayed, there was less suppression of flowering and fruit set. Ammonium thiosulfate and Endothal increased flowering but decreased fruit set compared to a control. Gramoxone suppressed flowering and fruit set. In another set of experiments, gibberellic acid (GA) treatments were evaluated to suppress FBI in "off" or light crop years. The GA4+7 treatments suppressed return bloom of both spur and lateral flowers more than the GA3 treatments. The effectiveness of GA declined with delayed application. Both GA treatments reduced lateral flowering the most on the basal 1/3 of the shoot. In a four year study, apple trees were thinned to one fruit per flowering cluster every year from 1997 to 2000. Other trees were thinned to zero fruit or two fruit per flowering cluster in alternate years from 1997 to 2000. Trees thinned to one fruit per flowering cluster had moderate flowering and fruit set the following year. Trees thinned to two fruit per flowering cluster had very little to no flowering the following year. Trees thinned to zero fruit per flowering cluster had a "snowball" bloom the following year. Trees that were alternately thinned to two or zero fruit per flowering cluster were in a biennial bearing situation. / Ph. D.

Gap junctions, comprised of connexin proteins, are essential for direct intercellular electrical, metabolic, and immunological coupling. Connexin43 (Cx43, gene name GJA1) is the most ubiquitously expressed gap junction protein, and Cx43 gap junctions are altered in pathological states including cardiac disease and cancer. The GJA1 mRNA undergoes alternative translation initiation to yield a truncated Cx43 isoform, GJA1-20k, that can regulate gap junction formation. Using epithelial-mesenchymal transition (EMT) as a cellular model of gap junction remodeling, we have demonstrated altered translation initiation of Gja1 as a mechanism by which cellular Cx43 gap junctions can be dynamically regulated. Suppression of Gja1 alternative translation is necessary for Cx43 gap junction loss, and stable expression of GJA1-20k rescues gap junction formation during EMT. To identify regulatory factors acting on the Gja1 mRNA, an MS2 RNA aptamer tagging system was adapted to isolate Gja1 with associated RNA binding proteins. We find the RNA binding protein IMP1 is sensitive to hypoxic stress and complexes with Gja1 mRNA, where it is necessary for alternative translation to generate GJA1-20k. We have demonstrated alterations in translation initiation of the Gja1 mRNA as a critical mechanism by which cells modulate Cx43 gap junctional coupling in changing conditions and identified a novel regulator of this process in mammalian cells. / Doctor of Philosophy / Communication between cells is necessary for healthy function of organs throughout the body. Gap junctions form conduits through which signals can pass directly between neighboring cells. Many diseases, including cancer and heart disease, involve disturbances in gap junction communication. Connexin proteins are the building blocks of gap junctions, and it was recently demonstrated that smaller fragments of connexins are synthesized by cells by a poorly understood process called alternative translation. Importantly, levels of these connexins fragments can alter gap junction formation. We have used mammalian cells to delineate the mechanism by which this alternative protein translation regulates gap junction formation and generated insight into how such protein synthesis is dynamically regulated. Harnessing this knowledge will inform development of new therapeutics inducing alternative translation to rescue gap junctions, and restore normal communication in pathological conditions.

Despite successful learning in the training environment, acquired skills are often not translated back to the job. Past research on training transfer has tended to measure the construct inconsistently and often disregarded its multi-faceted nature. In an effort to better investigate the determinants of successful transfer, the two temporal facets of training transfer, initiation and maintenance, were examined to evaluate their relationships with the trainee characteristics of goal orientation and self-efficacy. It was hypothesized that initiation mediates the relationship between goal orientation and maintenance, and that the relationship between performance goal orientation and initiation is moderated by self-efficacy. Participants were recruited from undergraduate psychology courses and trained on multiple-choice test-taking strategies. Results failed to support the main effect, moderation, or mediation hypotheses, although they support the contention that transfer is a multi-dimensional construct. The findings indicate that goal orientation and initiation may both best be conceived as predictors of transfer maintenance and interact to affect transfer behavior. The findings illustrate the value of examining individual difference variables in the prediction of training transfer. / Master of Science

Dans cette thèse, je me suis proposé de chercher à comprendre, en m'appuyant sur une étude critique d'écrits coloniaux et ethnographiques, comment se sont constituées les classifications des populations africaines à partir de présupposés raciologiques et les identifications réifiantes dans une région d'Afrique de l'ouest où le « mélange des races» avait frappé les premiers observateurs. Dans le même temps où ces représentations synchroniques s'imposaient, la conquête et l'administration coloniale ont contraint à des changements au niveau de l'activité productrice et du pouvoir ettransformé la vie collective et l'univers imaginaire et religieux. Une étude de terrain dans et autour du village de Sara situé dans la boucle du Mouhoun complète l'étude des écrits coloniaux et permet de mettre en évidence l'existence de réseaux de lignages au fondement de l'organisation sociale et politique, des distinctions qui en sont issues entre les Bwaba et les «étrangers », ce qui semble être à l'origine d'un sentiment d'appartenance à un groupe humain géographiquement indéfini, en Bwamu « Bwabawa .» Cette étude confirme également l'existence des transformations qui sont apparues pendantet après la colonisation. / Based on analysis of colonial and ethnographic studies, I tried to understand in this thesis how established the classifications of African populations from racial presumptions and reifying identifications in a region of western Africa where the "mixture of races" had struck the first observers. As these synchronous representations stood out, the conquest and the colonial administration forced changes to the productivity and to the existing power, transforming the people’s collective lives and their spiritual and religious worlds. A ground study in and around Sara's village, located in the loop of Mouhoun, completes the study of the colonial papers and highlights the existence of ethnic lineages in forming social and political structure, making distinctions between the Bwaba and the "foreigners", what seems to be at the origin of a feeling ofmembership of a geographically undefined human group, in Bwamu "Bwabawa.» This study also confirms the existence of transformations which appeared during and after colonization.

Durant ma thèse, j’ai étudié l’implication d’un variant du récepteur aux œstrogènes α, ERα36, dans l’initiation et la progression du cancer du sein. Au laboratoire, son expression dans les cancers testiculaires avait été montrée comme étant inductrice de la prolifération cellulaire in vitro et in vivo après une exposition à un mélange de polluants environnementaux, considérés comme perturbateurs endocriniens oestrogéno-mimétique : les alkylphénols. Une analyse rétrospective d’échantillons de tumeurs mammaires a montré, par la modélisation de réseaux d’interactions géniques, que l’expression d’ERα36 était corrélée avec l’expression de marqueurs de migration cellulaire, caractéristiques de la progression tumorale. La surexpression d’ERα36 par transfection in vitro et dans un modèle unique de souris Knocked In exprimant ERalpha36 dans la glande mammaire ont montré qu’ERα36 est suffisant pour altérer le phénotype épithélial des cellules mammaires saines. Une exposition aux alkylphénols qui stimulent son expression endogène accentue les altérations cellulaires observées et contribue à l’acquisition transgénérationnelle de propriétés relatives à une transformation tumorale. Les analyses de ce projet pluridisciplinaire se sont appuyées sur des expertises biologiques, mathématiques et bioinformatiques et ont permis de mettre en évidence pour la première fois le rôle potentiel d’ERα36 dans l’initiation tumorale et de confirmer son implication dans la progression du cancer du sein. Enfin, nous avons montré que l’exposition à des doses environnementales d’alkylphénols lors de la période de périnatalité peut conduire à une modification transgénérationnelle de la différenciation de la glande mammaire sous le contrôle d’ERα36 et ainsi augmenter le risque de cancer mammaire / This work was dedicated to study how a variant of estrogen receptor α, ERα36, acts in initiation and progression of breast cancer. In the laboratory, his expression in testicular cancer was shown to stimulate cell proliferation in vitro and in vivo after environmental pollutant exposure. The compounds studied, the alkylphenols, are endocrine disruptors, interfering with normal estrogen signaling. Gene interaction network modelling from retrospective analysis of breast cancer samples showed that ERα36 expression was correlated with the expression of cell migration markers, typical of tumor progression. In vitro ERα36 overexpression and in a unique mouse Knocked In model, expressing ERα36 in the mammary gland, showed that ERα36 is sufficient to alter epithelial phenotype of normal breast cells. Alkylphenols exposure, that stimulated ERα36 endogenous expression, increased cellular alterations and contributed to transgenerational acquisition of properties related to neoplastic transformation. Analysis of this multidisciplinary project were based on biological expertise, mathematics and bioinformatic tools. These results enabled to highlight for the first time the potential role of ERα36 in tumor initiation and confirmed his involvement in breast cancer progression. Finally, we showed that exposure to environmental doses of alkylphenols during the perinatal period can lead to transgenerational modification of mammary gland differentiation under ERα36 control and eventually may increase breast cancer risk

Le rayonnement ultraviolet (UV) émis par le soleil et qui atteint la peau de chaque individu est composé majoritairement de photons UVA (λ de 315 à 400 nm), le reste (5 à 10 %) étant composé d’UVB les plus longs (λ de 300 à 315 nm), car les radiations de longueur d’onde 300nm, c’est-à-dire les plus toxiques en terme de santé humaine, sont absorbées par la couche d’ozone stratosphérique. Contrairement aux UVB, les radiations UVA sont faiblement absorbées par l’ADN et de fait, génèrent peu de dimères cyclobutaniques de pyrimidines. Néanmoins, un des problèmes majeurs posés par une exposition aux UVA tient à ce que ce rayonnement excite certains composés endogènes photosensibles, inducteurs de la production d’espèces réactives de l’oxygène (ROS) qui peuvent alors endommager les composants cellulaires tels que les lipides,les acides nucléiques et les protéines. De ce fait, si les UVB restent le facteur étiologique majeur contribuant à la cancérogenèse cutanée photoinduite, un rôle des UVA, via la production de ROS, semble également émerger. Des précédents travaux obtenus au laboratoire ont montré que le rayonnement UVA ralentit la réplication de l’ADN, indépendamment de l’activation des points de contrôle du cycle cellulaire. Les auteurs ont émis l’hypothèse que les UVA, via l’oxydation des protéines, pouvaient altérer la machinerie de réplication. Mon travail de thèse a donc consisté à tenter de préciser le mécanisme qui gouverne ce retard de la réplication de l’ADN induit par les UVA dans les cellules de mammifères.Pour étudier au niveau moléculaire les effets des UVA sur la réplication, nous avons tout d’abord mis en place et utilisé au laboratoire la technique du peignage moléculaire (DNA combing) qui permet de mesurer divers paramètres de la réplication. Ainsi, nous montrons que le rayonnement UVA inhibe immédiatement et transitoirement les vitesses de fourches alors que l’inhibition sur l’initiation des origines est plus prolongée. Dans le cadre d’une collaboration, nous montrons également que les radiations UVA induisent une diminution modeste et transitoire du pool de dNTPs intracellulaires. La complémentation en ribonucléosides ne semble pas suffisante pour restaurer une vélocité normale de fourches immédiatement après UVA, ni la réplication dans sa totalité. En parallèle, nous observons l’oxydation réversible de la sous-unité R1 de la ribonucléotide réductase impliquée dans la biosynthèse des dNTPs. Bien que cette oxydation ne puisse expliquer la baisse transitoire du pool de nucléotides après UVA, nous ne pouvons pas exclure que d’autres formes d’oxydation de la RNR puissent affecter son activité.La présence d’azide de sodium (NaN3) au cours de l’irradiation UVA prévient le retard réplicatif, limite l’oxydation de la sous-unité R1 et la diminution du pool de dNTPs, ce qui démontre que ce retard de réplication est totalement dépendant des ROS, principalement de l’oxygène singulet généré pendant l’irradiation.L’ensemble de nos résultats indiquent que les UVA affectent le processus de réplication en modifiant non seulement la vélocité des fourches mais également l’initiation des origines de réplication. Puisqu’une perturbation de la réplication est une cause majeure d’instabilité génétique, il reste à déterminer si, dans nos conditions expérimentales, les radiations UVAfavorisent cette instabilité. Enfin, nous pensons que la ou les cibles des ROS induites par les UVA sont essentiellement cytosoliques et que le mécanisme conduisant à l’inhibition de la réplication n’est pas spécifique de ces ROS mais pourrait s’observer en utilisant d’autres types de stress oxydant. / The solar UV radiation that reaches the earth’s surface is composed of 10 % UVB (280–320 nm) and 90 % UVA (320–400 nm) the main toxic radiations (wavelengths below 300 nm) being blocked by the stratospheric ozone. Unlike UVB, the UVA component of solar radiation is weakly absorbed by DNA. Nevertheless, one of major problems due to UVA exposure is the production of reactive oxygen species (ROS) through the interaction with endogenous and exogenous chromophores. These ROS cause damage to DNA, lipids and proteins. Even if UVB remains the major etiological factor known to be implicated in photoinduced cutaneous carcinogenesis, a novel role for UVA via the production of ROS seems to emerge. In our lab, previous works have provided evidence that exposure of mammalian cells to UVA-induced ROS led to delayed S-phase and reduced DNA synthesis, by a yet unknown process, which does not require a functional DNA damage checkpoint response, despite ATM-, ATR-, p38-dependent pathways activation. The authors proposed that inhibition of DNA replication is due to impaired replication fork progression and/or origins activation, as a consequence of UVA-induced oxidative damage to proteins rather than to DNA. The project for my PhD thesis is to better understand the mechanism underlying this UVA-induced slowdown of DNA replication in human cells.To study at the molecular level the effects of UVA on DNA replication, we used the DNA combing methodology. This technique allows measurement of the fork velocity and of the origins density. We show that UVA-induced ROS inhibit immediately after irradiation, but transiently, the progression of replication forks, while the inhibition on the initiation of originslasts longer. By HPLC-MS, we show that UVA radiation induces a moderate and transient decrease of the level of each intracellular dNTP. The supply of ribonucleosides doesn’t seem to be sufficient to restore neither a normal forks velocity immediately post-UVA nor the overall slowdown of DNA replication. In addition, we observe a reversible oxidation of the subunit R1 of ribonucleotide reductase, an enzyme which is involved in dNTPs biosynthesis. This oxidation cannot explain the transient reduction of dNTPs pool after UVA exposure, but other types of RNR oxidative modification could affect its activity. During UVA irradiation, the presence of the antioxidant sodium azide (NaN3) prevents the delay of DNA replication, limits the oxidation of the subunit R1 and the decrease of dNTPs pool. These results strongly suggest that the slowdown of DNA replication totally depends on ROS, in particular on singlet oxygen production induced by UVA.Altogether, our data indicate that UVA irradiation affects the process of DNA replication by modifying the forks velocity and the activation of origins. As DNA replication impairment is a major cause of genetic instability, it is of importance to determine if UVA irradiation leads to this instability in our experimental conditions. Finally, we suspect that the target of UVAinduced ROS is essentially cytosolic and that the mechanism driving the inhibition of replication is not specific of UVA-induced ROS, but could be also observed with other types of oxidative stress.

Les facteurs environnementaux, tels que les pesticides, peuvent induire des changements phénotypiques dans une variété d'organisme incluant les mammifères. Nous avons étudié chez la souris les effets d'un pesticide largement utilisé, l'atrazine (ATZ), sur la méiose, une étape clé du processus de spermatogenèse. L'utilisation des méthodes de puces à ADN (Gene-Chip) et de séquençage de chromatine immunoprécipité (ChIP-seq) nous a permis de mettre en évidence l'effet de l'ATZ sur une variété de fonctions cellulaires, incluant l'activité GTPase, la fonction mitochondriale et le métabolisme des hormones stéroïdes. De plus, les souris traitées présentent un enrichissement des marques d'histone H3K4me3 au niveau des régions de forte recombinaison (sites de cassures double brin) de gènes très long et une réduction de ces mêmes marques au niveau des régions pseudo-autosomal du chromosome X. Nos données démontrent que l'exposition à l'ATZ interfère avec le déroulement normal de la méiose, ceci affectant la production des spermatozoïdes. Nous avons trouvé que les marques H3K4me3, chez la souris mâle, sont largement affectées par l'ATZ grâce à l'utilisation de technique de séquençage du génome entier. La reprogrammation embryonnaire nécessite l'action coordonnée d'un grand nombre de gène et de facteurs épigénétiques afin de permettre la transition de cellules somatique en cellules germinales. Les modifications épigénétiques imposées pendant la transition des cellules somatiques en cellules germinales et affectées par des expositions nocives, peuvent être héritées et transmises aux générations suivantes via les gamètes. Dans cette étude, nous avons examiné l'héritage des histones modifié aux générations suivantes. Nous avons exposés des femelles gestantes CD1 non consanguines à l'ATZ et les mâles issus de ces femelles ont été croisés pendant trois générations avec des femelles non traitées. Nous avons démontré ici que l'exposition à l'ATZ réduit le nombre de spermatozoïdes sans affecter la morphologie cellulaire ou la proportion des différents types cellulaires constituant l'épithélium séminifère chez les individus issus de la 3ème génération après traitement. Beaucoup de gènes associés avec la réparation de l'ADN, la reproduction et les fonctions mitochondriales sont dérégulés chez les mâles issus de la 3ème génération après traitement. De façon importante, l'exposition à l'ATZ change dramatiquement l'initiation de la transcription, l'épissage et la polyadénylation alternative des ARN. Nous avons aussi observé chez les mâles F3 issus de souris traitées à l'ATZ une altération de la localisation des marques H3K4me3 dans le promoteur de gène associé à la régulation de processus métaboliques cellulaires, à la régulation de la transcription et à la mitose. Les changements de localisation des marques H3K4me3 chez les mâles F3 issus de souris traitées à l'ATZ correspondent à des changements de la localisation de ces marques au niveau de gènes impliqués dans la différenciation des cellules de type souche de la génération F1.Nos données suggèrent que l'héritage transgénérationnel est permis grâce à de multiples voies et repose sur le statut épigénétique de gènes impliqués dans la différenciation des cellules de type souches tels que Pou5f1 et Sox2, l'action des facteurs de transcription et la rétention d'histones dans le sperme. / Environmental factors such as pesticides can cause phenotypic changes in various organisms, including mammals. We studied the effects of the widely used herbicide atrazine (ATZ) on meiosis, a key step of gametogenesis, in male mice. We demonstrate that exposure to ATZ reduces testosterone levels and the number of spermatozoa in the epididymis and delays meiosis. Using Gene-Chip and ChIP-Seq analysis of H3K4me3 marks, we found that a broad range of cellular functions, including GTPase activity, mitochondrial function and steroid-hormone metabolism, are affected by ATZ. Furthermore, treated mice display enriched histone H3K4me3 marks in regions of strong recombination (double-strand break sites), within very large genes and reduced marks in the pseudoautosomal region of X chromosome. Our data demonstrate that atrazine exposure interferes with normal meiosis, which affects spermatozoa production.We found that the H3K4me3 marks in male mice are broadly affected by the widely used herbicide atrazine with genome wide ChIP-sequencing. Embryonic reprogramming requires the coordinated action of many genes and epigenetic factors to perform somatic to germline transition. The epigenetic modifications imposed during somatic to germline transition and affected by harmful exposure can be inherited and transferred to subsequent generations via the gametes. In this study, we examine the inheritance of altered histone modifications by subsequent generations. We exposed pregnant outbred CD1 female mice to the widely used herbicide atrazine (ATZ), and the male progeny were crossed for three generations with untreated females. We demonstrate here that exposure to ATZ reduces the number of spermatozoa without changing the cell morphology or types in testis tissue in the third generation after treatment. Many genes associated with DNA repair, reproduction and mitochondrial function became dysregulated in the third generation (F3) of males after treatment. Importantly, exposure to ATZ dramatically changes the transcription initiation, splicing and alternative polyadenylation of RNA. We also observed altered occupancy of H3K4me3 markers in the F3 generation of ATZ-derived males in gene promoters associated with the regulation of cellular metabolic processes, transcriptional regulation and mitosis. The changes in H3K4me3 occupancy in F3 ATZ-derived males correspond to changes in the H3K4me3 occupancy of stem cell differentiation genes in the F1 generation. Our data suggest that transgenerational inheritance is accomplished through multiple pathways and relies on the epigenetic state of stem cell differentiation genes such as Pou5f1 and Sox2, transcription factor action and sperm histone retention.

<p>Initiation of protein synthesis in <i>E.coli </i>involves several steps, which lead to the formation of the first peptide bond. This process requires three initiation factors: IF1, IF2 and IF3. Using a novel technique of combined light scattering and stopped-flow, we elucidated the importance of IF2•GTP conformation for the recruitment of 50S to 30S pre-initiation complex. Moreover, GTP hydrolysis is essential for IF2 release and later binding of ternary complex. Interestingly, a switch in IF2 affinity to G-nucleotides is induced during 30S pre-initiation complexes formation. </p><p>We found that IF1, previously with unknown functions in vitro, increases the rate of naked 70S dissociation by a factor 80 and acts as a fidelity factor in preventing 70S formation containing elongator tRNA instead of fMet-tRNA^fMet. We showed that RRF/EFG/IF3 split both naked and post-termination complexes while IF1/IF3 split only naked ribosomes. The mechanisms of action of RRF/ EFG, the order of their binding to 70S, as well as, the three different conformation of EF-G on the ribosomes are emphasized. Interestingly, 70S formation rate is dependent on the concentration of IF3 and not linear with 50S subunits concentration. We demonstrated that the rate-limiting step in 70S formation is IF3 dissociation from 30S complexes.</p><p>The interplay between initiation factors in the rate and accuracy of protein synthesis was thoroughly studied. Using fMet-tRNA^fMet (initiator tRNA), Met-tRNA^fMet(non-formylated initiator tRNA) and Phe-tRNA^Phe (elongator tRNA), we showed that the major player in the accuracy is IF2 through recognizing the formyl group on fMet-tRNA^fMet, while IF3 acts by increasing both the on- and off-rate of tRNA from 30S pre-initiation complexes.</p><p>Collectively, these novel results describe a comprehensive model of initiation of protein synthesis. In this model, initiation factors increase the rate of fMet-tRNA^fMet binding to 30S subunits, subsequently; the stabilization of fMet-tRNA^fMet by IF2 increases the rate of IF3 dissociation. Later, IF2 in GTP conformation allows 50S docking to 30S pre-initiation complex free of IF3 followed by GTP hydrolysis allowing IF2 release for ternary complex to bind and start elongation of protein synthesis. </p>

Initiation of protein synthesis in E.coli involves several steps, which lead to the formation of the first peptide bond. This process requires three initiation factors: IF1, IF2 and IF3. Using a novel technique of combined light scattering and stopped-flow, we elucidated the importance of IF2•GTP conformation for the recruitment of 50S to 30S pre-initiation complex. Moreover, GTP hydrolysis is essential for IF2 release and later binding of ternary complex. Interestingly, a switch in IF2 affinity to G-nucleotides is induced during 30S pre-initiation complexes formation. We found that IF1, previously with unknown functions in vitro, increases the rate of naked 70S dissociation by a factor 80 and acts as a fidelity factor in preventing 70S formation containing elongator tRNA instead of fMet-tRNAfMet. We showed that RRF/EFG/IF3 split both naked and post-termination complexes while IF1/IF3 split only naked ribosomes. The mechanisms of action of RRF/ EFG, the order of their binding to 70S, as well as, the three different conformation of EF-G on the ribosomes are emphasized. Interestingly, 70S formation rate is dependent on the concentration of IF3 and not linear with 50S subunits concentration. We demonstrated that the rate-limiting step in 70S formation is IF3 dissociation from 30S complexes. The interplay between initiation factors in the rate and accuracy of protein synthesis was thoroughly studied. Using fMet-tRNAfMet (initiator tRNA), Met-tRNAfMet (non-formylated initiator tRNA) and Phe-tRNAPhe (elongator tRNA), we showed that the major player in the accuracy is IF2 through recognizing the formyl group on fMet-tRNAfMet, while IF3 acts by increasing both the on- and off-rate of tRNA from 30S pre-initiation complexes. Collectively, these novel results describe a comprehensive model of initiation of protein synthesis. In this model, initiation factors increase the rate of fMet-tRNAfMet binding to 30S subunits, subsequently; the stabilization of fMet-tRNAfMet by IF2 increases the rate of IF3 dissociation. Later, IF2 in GTP conformation allows 50S docking to 30S pre-initiation complex free of IF3 followed by GTP hydrolysis allowing IF2 release for ternary complex to bind and start elongation of protein synthesis.