Protein biosynthesis is a core process in all living organisms. Assembly of the protein chain from aminoacids is catalysed by the ribosome, ancient and extremely complex macromolecular machine. Several different classes of accessory molecules are involved in translation, and one set of them, called translational GTPases (trGTPases), was in the focus of this work. In this thesis properties of two trGTPases– EF-G and eRF3 - were studied by means of direct biochemical experiments. EF-G is a bacterial trGTPase involved in two steps of translation: translocation and ribosomal recycling. Translocation is a process of the ribosomal movement along the mRNA, and recycling as the step when upon completion of the protein ribosome is released from the mRNA via splitting in two ribosomal subunits. We found that off the ribosome EF-G has similar affinities to GDP and GTP, and thus given the predominance of the latter in the cell, EF-G should be present mostly in the complex with GTP. However, binding to the ribosome increases factors affinity to GTP drastically, ensuring that it is in the GTP-bound state. GDP can not promote neither translocation, not recycling, and GDPNP can not promote recycling. It can, however, promote translocation, but in so doing it results in an intermediate ribosomal state and translocation process can be reversed by addition of GDP, which is not the case for the EF-G•GTP-catalyzed reaction. The second trGTPase we investigated is eukaryotic termination factor eRF3. This protein together with another factor, eRF1, is involved translation termination, which is release of the synthesized protein from the ribosome. We demonstrateed, that eRF3 alone has basically no propensity to bind GTP and thus resides in the GDP-bound state. Complex formation between eRF1 and eRF3 promotes GTP binding by the latter, resulting in the formation of the ternary complex eRF1•eRF3•GTP, which in turn is catalyzing the termination event. Experimental investigations of trGTPases where rationalized within a generalized thermodynamical framework, accommoding the existent experimental observations, both structural and biochemical.

Signal responsive transcriptional control in bacteria is mediated through both specific and global regulatory circuits to attune promoter output to prevailing conditions. Divergent transcription of a regulatory gene and a cognate promoter under its control provides an opportunity for interplay between transcription dependent on RNA polymerases utilizing various σ-factors, each of which programs the holoenzyme to recognize different classes of promoters. The work presented in this thesis analyses the consequences and mechanisms behind interplay between σ54- and σ70-dependent transcription within the dmp-system of Pseudomonas sp. CF600. The dmp-system confers the ability to grow at the expense of (methyl)phenols and is controlled by two promoters that drive non-overlapping divergent transcription from a common intergenic region: i) the σ54-Po promoter, which controls an operon encoding a suit of specialized catabolic enzymes, and ii) the σ70-Pr promoter, which controls production of the aromatic sensor DmpR - a mechano-activator whose transcription-promoting activity is obligatory for activity of the σ54-Po promoter. The σ54-Po promoter and its dependence on two non-classical transcriptional regulators - the alarmone ppGpp and its co-factor DksA that directly target RNA polymerase - are the focus of the first part of the thesis. These studies utilized ppGpp and DksA deficient strains, mutant RNA polymerases that bypass the need for ppGpp and DksA, reconstituted in vitro transcription systems, and a series of DmpR-regulated hybrid σ54-promoters with different affinities for σ54-RNA polymerase, together with analysis of protein levels of key transcriptional components. Collectively with previous work, these studies provide the experimental support for a robust but purely passive mechanism for ppGpp and DksA global regulation of σ54-transcription, which is likely to also be pertinent for transcription mediated via any alternative σ-factor (Papers I-III). The second part of the thesis focuses on additional roles of ppGpp and DksA through their direct and indirect effects on the activity of the σ70-Pr promoter. These studies unexpectedly revealed that the σ70-Pr promoter is regulated by a novel mechanism in which σ54-RNA polymerase occupancy and activity at the σ54-Po promoter stimulates σ70-Pr output. Evidence is presented that ppGpp and DksA, through DmpR levels, control a feed forward loop to reinforce silence of the σ54-Po promoter under high energy conditions with robust transcription from σ54-Po when the catabolic enzymes are needed. The interplay outlined above effectively places a σ70-dependent promoter under dual control of two forms of RNA polymerases, and also makes it subservient to regulatory signals that elicit activity of σ54-RNA polymerase. The possibility that such dual sensitivity may be a prevalent, but previously unappreciated, mechanism by which bacteria integrate diverse and/or conflicting signals to gain appropriate transcriptional control is discussed.

Mitochondria are eukaryotic cellular organelles responsible for power-generation, believed to have come into existence by an endo-symbiontic event where a bacterial cell was incorporated by an un-specified "proto-eukaryote". Phylogenetic analysis have shown that the mitochondrial ancestor was most related to present-day alpha-proteobacteria, although the exact nature of the mitochondrial progenitor remains disputed. In this work, I have used phylogenetic and other methods to investigate the identity of the organism giving rise to mitochondria, by analysing the evolutionary history of select proteins, the events where they have been transfered to the eukaryotic nucleus, and the time-point of mitochondrial establishment. In addition, a search for mitochondrially related organisms in the ocean metagenome was performed, in the hope that something more related to the mitochondrial progenitor than anything previously identified could be found. Previous analysis have shown that a large fraction of mitochondrial proteins does indeed trace their descent to the alpha-proteobacteria, but I found that the amino-acyl tRNA-synthetases display more general bacterial descent, making it likely that these proteins are of a different origin from the mitochondria themselves. While the synthetases are encoded on the nuclear genome, most mitochondria still posses most of the tRNA on their own genomes. In the cases where the tRNA has been lost from the mitochondrial genome, I found that the probability of loss correspond to the evolutionary history of their synthetase. The ocean metagenome represents an order of magnitude more data than previously available, making it suitable for improving the analyses dealing with mitochondrial placement. This large of amount of data was utilised to improve the phylogenetic analyses, showing that previous works might have suffered from artefacts inflating the support for placement of mitochondria with a specific alpha-proteobacterial group. Eukaryotic/mitochondrial radiation was shown to be as old, or older, than radiation of extant alpha-proteobacteria, casting doubt on previous analysis identifying a specific alpha-proteobacterial group as the mitochondrial ancestor.

A common characteristic in neurodegenerative diseases of the brain is death of specific neuronal populations. The lack of neuron proliferation and axon extension in most parts of the central nervous system leads to chronic loss of neurons in the case of injury or disease. Therefore it is essential to identify signals involved in neurogenesis and neuronal survival. A favorable model in which to study these events is the olfactory sensory neurons in the main olfactory epithelium and their target in the glomeruli of the olfactory bulb. In spite of constant regeneration, each olfactory sensory neuron maintain expression of one particular odorant receptor and the specificity of their axonal projections to the glomeruli. Most mammals also have an accessory olfactory system consisting of the sensory neurons in the vomeronasal epithelium and their target area the accessory olfactory bulb. Differential expression of receptors and other genes divides the olfactory and vomeronasal epithelium into zones, but the function and mechanisms underlying the establishment of these zones are still elusive. We identified four genes with graded expression patterns that correlated with the zones of the olfactory epithelium. One of the identified genes encodes a retinoic acid synthesizing enzyme, RALDH-2. We showed that RALDH-2 was expressed in a gradient in cells of the lamina propria underneath the olfactory epithelium, suggesting a possible retinoic acid regulation of zonally expressed genes in the olfactory epithelium. To investigate the role of retinoic acid in the olfactory systems, we generated a transgenic mouse strain that selectively expressed a dominant negative retinoic acid receptor in mature olfactory and vomeronasal neurons. We found that subsequent to the establishment of axonal projections, the neurons of both olfactory systems died prematurely by retrograde caspase-3 activation. In the main olfactory system the onset of apoptosis was associated with the appearance of incorrect heterogenous glomeruli with axons of more than one OR identity. Additionally, the activity regulated cell adhesion molecule kirrel-2 was down regulated suggesting an additional regulation of this gene by retinoic acid. Deficient retinoic acid signaling in olfactory sensory neurons could thus induce apoptosis by changing the parameters for axonal competition by neural activity and kirrel-2 expression. We found evidence for a selective neuronal death in the accessory olfactory system of the dnRAR mice, where only vomeronasal sensory neurons belonging to the basal zone died by retrograde caspase-3 activation. This implies that the two populations of sensory neurons in the vomeronasal epithelium differently depend on retinoic acid for their survival.

The presence and synthesis of transfer RNA (tRNA) is highly conserved in all organisms and a lot of genetic material is dedicated to its synthesis. tRNA contains a large number of modified nucleosides and several diverse functions have been found but much about their function is still unknown. By using a novel frameshifting system to select for tRNA modification mutants, new mutations were isolated and subsequently analyzed. This thesis examines the synthesis and function of a subset of tRNA modifications that have a sulfur (thio) -group as part of the modification. The isc operon encodes for proteins synthesizing iron sulfur centers ([Fe-S]) that are a part of the active site of many key enzymes in the cell and the thiolated nucleosides are dependant on a functional iron sulfur gene (iscS) for their synthesis. By studying thiolated tRNA it is not only possible to learn more about the synthesis of the modifications themselves, but also about the synthesis of [Fe-S] clusters. Based on an analysis of mutations in three of the isc operon genes (iscS, iscU, and iscA), a two-model pathway is proposed for the synthesis of Salmonella enterica Serovar Typhimurium thiolated tRNA modifications. The interactions of IscS with other proteins in the tRNA modification thiolation pathways suggest a more complex sulfur relay than had previously been envisioned. Some of the specificities and the effect of an iscA mutant on the levels of tRNA modifications lead to an examination of the role of IscA in [Fe-S] formation and its importance for tRNA modifications.

The proteasome is a major non-lysosomal proteolytic complex present in eukaryotic cells and has a central role in regulating many protein levels. The complex has been shown to participate in various intracellular pathways including cell cycle regulation or quality control of newly synthesized proteins and many other key pathways. This amazing range of substrates would not be possible without the help of regulators that are able to bind to the 20S proteasome and modulate its activity. Among those, the PA700 or 19S regulator and the PA28 family are the best characterized in higher eukaryotes. The 19S regulatory particle is involved in the recognition of ubiquitinated proteins, targeted for degradation by the proteasome. The PA28 (also termed 11S REG) family is composed of two members the PA28αβ and PA28γ. The function of PA28αβ is related to the adaptive immune response with a proposed contribution in MHC class I peptide presentation whereas the biological role PA28γ remains unknown. The main objectives of the laboratory, and subsequently of this thesis are to use Drosophila melanogaster model system and its advantages to better understand the precise contribution of these different activators in the regulation of the proteasome. Using genomic resources, a unique Drosophila PA28 member was identified, characterized and was shown to be a proteasome regulator with all the properties of PA28γ. Through site-directed mutagenesis we identified a functional nuclear localization signal in the homolog-specific insert region. Study of the promoter region revealed that transcription of Drosophila PA28γ (dPA28γ) gene is under control of DREF, a transcription factor involved in the regulation of genes related to DNA synthesis and cell proliferation. To confirm that dPA28γ has a role in cell cycle progression, the effect of removing dPA28γ from S2 cells was tested using RNA interference. Drosophila cells depleted of dPA28γ showed partial arrest in G1/S cell cycle transition confirming a conserved function between Drosophila and mammalian forms of PA28γ. Finally, characterization of the Dictyostelium regulator, an evolutionarily distant member of the PA28γ, was carried out using fluorogenic degradation assays. We are currently knocking-out the gene in order to determine the biological function of the activator. A second part of my work consisted in the generation of a Drosophila assay used to identify in vivo substrates of the 19S regulator, an assay system that has been originally engineered by Dantuma and coworkers in human cell lines. This was achieved by cloning of GFP behind a series of modified ubiquitins that create substrates degraded through different pathways involving the proteasome pathways. The last project of my thesis concerns the characterization of the mechanism for upregulation of proteasomal gene mRNA after MG132 (proteasome inhibitor) treatment. So far, we found that the 5´-UTR of the genes is responsible for this induction. We are now looking for the precise motif involved in this regulation.

My initial aim was a functional analysis of the conserved Op18/stathmin family of microtubule-regulators, which includes the ubiquitous cytosolic Op18 protein and the neural membrane-attached RB3 and SCG10 proteins. The solved X-ray structure has shown that these proteins form a complex with tubulin -heterodimers via two imperfect helical repeats, which result in two head-to-tail aligned heterodimers in a tandem-tubulin complex. We have analyzed GTP exchange and GTP hydrolysis at the two exchangeable GTP-binding sites (E-site) within the tandem-tubulin complex. A comparison of Op18, RB3 and SCG10 proteins indicates that Op18/Stathmin family proteins have evolved to maintain the two heterodimers in a configuration that restrains the otherwise potent GTPase productive interactions facilitated by the head-to-head alignment of heterodimers in protofilaments. We concluded from these studies that tubulin heterodimers in complex with Op18/stathmin family members are subject to allosteric effects that prevent futile cycles of GTP hydrolysis. To understand the significance of the large differences in tubulin affinity of Op18, RB3 and SCG10, we have fused each of the heterodimer-binding regions of these three proteins with the CD2 cell-surface protein to generate confined plasma membrane localization of the resulting CD2 chimeras. We showed that, in contrast to CD2-Op18, both the CD2-SCG10 and CD2-RB3 chimeras sequester tubulin at the plasma membrane, which results in >35% reduction of cytosolic tubulin heterodimer levels. However, all three CD2-chimeras, including the tubulin sequestration-incompetent CD2-Op18, destabilize interphase microtubules. Given that microtubules are in extensive contact with the plasma membrane during the interphase, these findings indicate that Op18-like proteins have the potential to destabilize microtubules by both sequestration and direct interaction with microtubules. Sm16/SmSLP (Stathmin-Like Protein) has been identified as a protein released during skin penetration of the Schistosoma mansoni parasite. This protein has been ascribed both anti-inflammatory activities and a functional similarity with the conserved cytosolic tubulin-binding protein stathmin/Op18. However, our studies refuted any functional similarity with stathmin/Op18 and we found instead that Sm16/SmSLP is a lipid bilayer binding protein that is taken up by cells through endocytosis. To study immuno-modulatory properties of Sm16/SmSLP, we designed an engineered version with decreased aggregation propensity, thus facilitating expression and purification of a soluble Sm16 /SmSLP protein from the eukaryotic organism Pichia pastoris. Determination of the hydrodynamic parameters revealed that both the recombinant and native Sm16/SmSLP is a ~9-subunits oligomer. The recombinant protein was found to have no effect on T lymphocyte activation, cell proliferation or the basal level of cytokine production of whole human blood or monocytic cells. Interestingly, however, recombinant Sm16 was found to potently inhibit the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and Poly(I:C). Since Sm16 specifically inhibits degradation of the IRAK1 signaling protein in LPS stimulated monocytes, it seems likely that inhibition is exerted proximal to the TLR-complex.

The Gram-positive human pathogen Listeria monocytogenes uses a wide range of virulence factors for its pathogenesis. The majority of its virulence genes are encoded on a 9-kb pathogenicity island and are controlled by the transcriptional activator PrfA. Expression of these genes is maximal at 37°C and minimal at 30°C in a mechanism involving an RNA thermosensor. This thesis brings up different aspects of RNA-mediated regulation, including regulatory RNA structures within coding mRNA controlling expression to 5-untranslated RNA (5´-UTR) that controls downstream genes (cis-acting) as well as small non-coding RNAs (ncRNAs) that bind other target RNA (trans-acting). We investigated the importance of the coding region of the prfA-mRNA for its expression. Various lengths of prfA-mRNA were fused with reporter genes. Our finding suggested that the first 20 codons of prfA-mRNA were essential for efficient translation in Listeria monocytogenes. Translation of the shorter constructs was shown to be reduced. The expression level showed an inverse correlation with the RNA secondary structure stability in the beginning of the coding region. Riboswitches have previously been known to control expression of their downstream mRNA in a cis-acting manner. A trans-acting S-adenosylmethionine-binding riboswitch termed SreA was identified in Listeria monocytogenes. It was found to control the expression of the virulence regulator PrfA, by binding to the prfA-UTR and thereby affecting its translation. We examined the RNA locus encoding different virulence factors in Listeria monocytogenes. Several of them were preceded by 5´-UTRs of various lengths. We speculate that these 5´-UTRs could control expression of the downstream mRNA, provided they are of sufficient length. These findings prompted us to examine where and when Listeria monocytogenes switches on gene expression. Tiling array was used to compare RNAs isolated from wild-type and mutant bacteria grown at different growth conditions. Antisense RNAs covering parts of or whole open-reading frames as well as 29 new ncRNAs were identified. Several novel riboswitches possibly functioning as upstream terminators were also found. My thesis work compiles together a variety of novel RNA-mediated gene regulatory entities. A first coordinated transcriptional map of Listeria monocytogenes has been set up. My work has also revealed that the expression of the virulence regulator PrfA is controlled at several levels, indicating the importance of both the 5´-UTR and the coding RNA for regulated expression.

The assembly of the ribosome is a complex process that needs to be highly efficient to support maximum growth. Although the individual subunits of the ribosome can be reconstituted in vitro, such a reaction is inefficient in comparison to the assembly rate in vivo. What differentiates the in vivo from the in vitro assembly is primarily the presence of ribosome assembly proteins. These are proteins that assist in the assembly of the ribosomal subunits but are not part of the mature ribosome. In bacteria, the ribosome assembly proteins include rRNA processing enzymes and rRNA/ribosomal protein (r-protein) modifying enzymes. One set of ribosome assembly proteins, the ribosome maturation factors, have been difficult to classify due to their differences in structure and their apparent lack of similarities with regard to function. As part of this thesis, the previously uncharacterized RimP (ribosome maturation) protein formerly known as P15A or YhbC, was studied. Deletion of the rimP gene affected the growth rate more severely at 44°C than at 37°C and 30°C. Polysome profile analysis revealed a decrease in the amount of translating ribosomes and a corresponding increase in the amount of free 50S and 30S ribosomal subunits. The disproportionate large increase in 50S relative to 30S subunits indicated a 30S assembly defect. RimP was shown to localize to the 30S ribosomal subunit, and an accumulation of 17S rRNA, a precursor to 16S rRNA, supports a role for RimP in 30S subunit maturation. The results from in vitro reconstitution experiments have given valuable insights in the assembly of the 30S subunit. By using a recently developed method, the role of ribosome maturation factors Era, RimM and RimP during in vitro reconstitutions of the 30S subunit was investigated. Era was found to increase the incorporation rate for most of the late binding r-proteins, while RimM and RimP had more specific effects. RimM increased the incorporation rate for r-proteins S19 and S9 and inhibited the incorporation of S13 and S12, whereas RimP increased the incorporation rate primarily for S12 and S5. A comparison of the ribosome maturation factors RimP and RbfA (ribosome binding factor A) revealed structural similarities between the N-terminal domain of RimP and the single domain of RbfA. RbfA is a 15 kDa protein that was found to high copy-suppress a dominant C23U 16S rRNA mutation giving rise to cold-sensitivity in E. coli. A number of chromosomal suppressor mutations that increased the growth rate of an rbfA null mutant were isolated. The five strongest suppressor mutations were localized to the rpsE gene, for r-protein S5 and resulted in amino acid substitutions in three positions: G87A, G87S, G91A, A127V and A127T. These alterations improved translation and the processing of 16S rRNA in the rbfA null mutant. Moreover, they also suppressed the slow growth of the C23U rRNA mutant at 30, 37 and 44°C. / Monteringen av ribosomen är en komplex process som måste vara effektiv för cellen skall kunna växa så fort som möjligt. Det är visat sedan tidigare att ribosomens två subenheter kan monteras ihop in vitro och sedan vara del av en ribosom som gungerar vid proteinsyntes, dock är den typen av rekonstrueringsreaktioner mycket ineffektiva i jämförelse med vad som krävs in vivo. Skillnaden mellan dessa två tillstånd är primärt in vivo-reaktionens närvaro av hjälpproteiner. Hjälpproteinerna assisterar monteringen av ribosomens subenheter men är själva inte en del av den färdiga ribosomen. Inom denna klass av proteiner återfinns proteiner som t.ex. processerar ribosomalt RNA och proteiner som modifierar ribosomalt RNA och ribosomala protein. En klass av hjälpproteiner, mognadsfaktorerna, har varit svåra att klassificera på grund av strukturella olikheter och en brist på funktionella likheter. En del i detta avhandlingsarbete var karaktäriseringen av den tidigare okända mognadsfaktorn RimP, tidigare kallad YhbC eller P15A. En deletion av rimP hade störst påverkan på tillväxthastigheten vid 44°C, men effekter kunde även ses vid 30°C och 37°C. En analys av den ribosomala statusen visade på en minskning av ribosomer aktiva i translation och en motsvarande ökning av fria 50S- och 30S-subenheter. Den oproportionerligt höga ökningen av fria 50S-subenheter, i relation till 30S-subenheter, indikerade att något var fel i monteringen av 30S-subenheten. RimP-proteinet återfanns lokaliserat till fria 30S-subenheter, och en ökning av omoget 16S ribosomalt RNA i en stam som saknar RimP stödjer dess roll i monteringen av 30S-subenheten. Rekonstrueringsexperiment In vitro har gett många värdefulla ledtrådar till hur 30S-subenheten monteras ihop. Genom att använda en nyligen utvecklad metod kunde vi undersöka hur mognadsfaktorerna Era, RimM och RimP påverkade monteringen av ribosomens 30S subenhet in vitro. Era ökade inkorporeringshastigheten av många av de ribosomala proteiner som inkorporeras sent i monteringen av 30S, medans RimM och RimP uppvisade mer specifika effekter. RimM ökade inkorporeringshastigheten för de ribosomala proteinerna S19 och S9, men dessutom inhiberade RimM inkorporeringen av de ribosomala proteinerna S13 och S12. RimP uppvisade den tydligaste effekten av de undersökta proteinerna genom att kraftigt öka 8 inkorporeringshastigheten för det ribosomala proteinet S12, och ökade även inkorporeringshastigheten för det ribosomala proteinet S5. En jämförelse av de två mognadsfaktorerna RbfA och RimP visade på strukturella likheter mellan RimP:s N-terminala domän och den enda domänen hos RbfA. RbfA är ett 15 kDa protein som upptäcktes som en hög-kopiesupressor av en dominant C23U-mutation i 16S ribosomalt RNA som leder till köld-känslighet hos E. coli. Ett antal kromosomala supressormutationer som ökade tillväxthastigheten för en mutant som saknar RbfA isolerades och de fem starkaste av dessa lokaliserades till rpsE genen som kodar för det ribosomala proteinet S5. Mutationerna gav upphov till aminosyra utbyten i tre positioner i S5: G87A, G87S, G91A, A127T och A127V. Förändringarna i S5 förbättrade translationen och processningen av 16S ribosomalt RNA i mutantensom saknar RbfA. Dessutom förbättrade mutationerna tillväxthastigheten hos C23U-mutanten vid 30, 37 och 44°C.

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