Tn7 and Tn7-like transposons are complex elements found in disparate environments and are responsible for mobilizing a wide variety of genes and forming pathogenicity/fitness islands. They are novel in their ability to recognize both a single site in the chromosome and specifically target transposition into mobile plasmids via dedicated TnsD and TnsE targeting proteins. TnsE recognizes mobile plasmids through an association with the processivity clamp and a 3′ recessed DNA end during conjugal replication. However, the mechanism for the specific recognition of 3′ recessed DNA ends remains unclear. Structural analyses of the C-terminal domain of TnsE identified a novel protein fold including a central V-shaped loop that toggles between two distinct conformations. The structure of a robust TnsE gain-of-function variant has this loop locked in a single conformation, suggesting that conformational flexibility regulates TnsE activity. Structure-based analysis of a series of TnsE variants relates transposition to DNA binding stability. Follow up studies of full length TnsE bound to DNA are in progress. / Thesis / Master of Science (MSc)

No description available.

<p>DNA replication is a fundamental process that must be precisely regulated to ensure timely and faithful transmission of genetic material for proliferation of all organisms. Replication initiation is regulated through a series of precisely timed protein–DNA and protein–protein interactions. In <em>Escherichia coli</em>, one regulatory mechanism of replication initiation occurs through SeqA binding to specific sequences within the <em>oriC</em>, resulting in origin sequestration. SeqA also plays a role in chromosome organization at the replication forks. Despite the functional importance of SeqA in <em>E. coli</em>, its DNA binding mechanism has remained elusive. The work described in this thesis has shown for the first time the minimal functional unit of SeqA that forms a high-affinity complex with DNA through the loss of symmetry. This is a novel observation that explains how SeqA can distinguish template versus newly replicated strand of DNA. We have also identified a protein–protein interaction surface that separates the roles of SeqA at the origin in sequestration and at the replication forks in chromosome organization. The final contribution of the thesis is in the exploration of SeqA functions in other bacterial species and demonstrating the structural and functional similarities between <em>Vibrio cholerae </em>SeqA and <em>E. coli </em>SeqA. Together our work has made a crucial connection between the structural organization of the protein and its functional ability to bind DNA.</p> / Doctor of Philosophy (PhD)

All living organisms must organize their genome so that it not only fits within the cell, but remains accessible for cellular processes. In bacteria, an arsenal of nucleoid-associated proteins contributes to chromosome condensation. A novel nucleoid-associated protein was recently discovered in actinobacteria, and is essential in Mycobacterium. It was classified as an integration host factor protein (IHF); however, it does not share sequence or structural homology with the well characterized Escherichia coli IHF. In this study, we characterize the structure and function of Streptomyces coelicolor IHF (sIHF). We have used a combination of biochemistry and structural biology to characterize the role of sIHF in DNA binding and DNA topology. We have solved crystal structures of sIHF bound to various double-stranded DNA substrates, and show that sIHF is able to contact DNA at multiple surfaces. Furthermore, sIHF inhibits the activity of TopA, impacting DNA topology in vitro. Our work demonstrates that sIHF is a novel nucleoid-associated protein with key roles in condensing DNA. We believe that sIHF performs its function by differentially using multiple nucleic-acid binding surfaces. Further characterization is required to confirm this hypothesis in vivo. Given that the Mycobacterium homolog of sIHF (mIHF) is essential, our studies lay the foundation to explore novel drug targets for Mycobacterium tuberculosis and Mycobacterium leprae. / Thesis / Master of Science (MSc) / Unconstrained, the bacterial genome exceeds the size of the cell by 1 000- 10 000 times; thus, compacting it into a condensed structure, known as the nucleoid, is essential for life. An arsenal of nucleoid-associated proteins contributes to this process. In this study, we characterize the structure and function of a novel nucleoid–associated protein from the soil dwelling organism Streptomyces coelicolor. We used a combination of genetics, biochemistry, and structural biology to characterize the role of this protein in DNA binding and nucleoid organization. Since this protein is also found in important human pathogens, this work lays the foundation to explore the use of nucleoid-associated proteins as antimicrobial drug targets.

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第25399号 / エネ博第478号 / 新制||エネ||89(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 森井 孝, 教授 片平 正人, 教授 佐川 尚 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第25400号 / エネ博第479号 / 新制||エネ||89(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 森井 孝, 教授 片平 正人, 教授 佐川 尚 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

No description available.

During the past decade the effects of macromolecular crowding on reaction pathways is gaining in prominence. The stress is to move out of the realms of ideal solution studies and make conceptual modifications that consider non-ideality as a variable in our calculations. In recent years it has been shown that molecular crowding exerts significant effects on all in vivo processes, from DNA conformational changes, protein folding to DNA-protein interactions, enzyme pathways and signalling pathways. Both thermodynamic as well as kinetic parameters vary by orders of magnitude in uncrowded buffer system as compared to those in the crowded cellular milieu. Ignoring these differences will restrict our knowledge of biology to a “model system” with few practical understandings. The recent expansion of the genome database has stimulated a study on numerous previously unknown proteins. This has whetted our thirst to model the cellular determinants in a more comprehensive manner. Intracellular extract would have been the ideal solution to re-create the cellular environment. However, studies conducted in this solution will be contaminated by interference with other biologically active molecule and relevant statistical data cannot be extracted out from it. Recent advances in methodologies to mimic the cellular crowding include use of inert macromolecules to reduce the volume occupancy of target molecules and the use of immobilization techniques to increase the surface density of molecules in a small volumetric region. The use of crowding agents often results in non-specific interaction and side-reactions like aggregation of the target molecules with the crowding agents themselves. Immobilization of one of the interacting partners reduces the probability of aggregation and precipitation of bio-macromolecules by restricting their degrees of freedom. Covalent linkage of molecules on solid support is used extensively in research for creating a homogeneous surface of bound molecules which can be interrogated for their reactivity. However, when it comes to biomolecules, direct immobilization on solid support or use of organic linkers often results in denaturation. The use of bio-affinity immobilization techniques can help us overcome this problem. Since mild conditions are needed to regenerate such a surface, it finds universal applicability as bio-memory chips. This thesis focuses on our attempts to design a physiologically viable immobilization technique for following rotein-protein/protein-DNA interactions. The work explores the mechanism for biological interactions related to transcription process in E. coli. Chapter 1 deals with the literary survey of the importance and effects of molecular crowding on biological reactions. It gives a brief history of the efforts been made so far by experimentalists, to mimic macromolecular crowding and the methods applied. The chapter tries to project an all-round perspective of the pros and cons of different immobilization techniques as a means to achieve a high surface density of molecules and the advancements so far. Chapter 2 deals with the detailed technicality and applicability of the Langmuir-Blodgett method. It discusses the rationale behind our developing this technique as an alternate means of bio-affinity immobilization, under physiologically compatible conditions. It then goes on to describe our efforts to follow the sequence-specific and sequential assembly process of a functional RNA polymerase enzyme with one immobilized partner and also explore the role of omega subunit of RNAP in the reconstitution pathway. This chapter uses the assembly process of a multi-subunit enzyme to evaluate the efficiency of the LB system as a universal two-dimensional scaffold to follow sequence-specific protein-ligand interaction. Chapter 3 discusses the application of LB technique to quantitatively evaluate the kinetics and thermodynamics of promoter-RNA polymerase interaction under conditions of reduced dimensionality. Here, we follow the interaction of T7A1 phage promoter with Escherichia coli RNA polymerase using our Langmuir-Blodgett technique. The changes in mechanistic pathway and trapping of kinetic intermediates are discussed in detail due to the imposed restriction in the degrees of freedom of the system. The sensitivity of this detection method is compared vis-a-vis conventional immobilization methods like SPR. This chapter firmly establishes the universal application of LB technique as a means to emulate molecular crowding and as a sensitive assay for studying the effects of such crowding on vital biological reaction pathway. Chapter 4 describes the mechanistic pathway for the physical binding of MsDps1 protein with long dsDNA in order to physically protect DNA during oxidative stress. The chapter describes in detail the mechanism of physical sequestering of non-specific DNA strands and compaction of the genome under conditions where a kinetic bottleneck has been applied. The data obtained is compared with results obtained in the previous chapter for the sequence-specific DNA-protein interaction in order to understand the difference in recognition process between regulatory and structural proteins binding to DNA. Chapter 5 deals with the evaluation of the σ-competition model in E. coli for three different sigma factors (all belonging to the σ-70 family). Here again, we have evaluated the kinetic and thermodynamic parameters governing the binding of core RNAP with its different sigma factors (σ70, σ32and σ38) and performed a comparative study for the binding of each sigma factor to its core using two different non-homogeneous immobilization techniques. The data has been analyzed globally to resolve the discrepancies associated with establishing the relative affinity of the different sigma factors for the same core RNA polymerase under physiological conditions. Chapter 6 summarizes the work presented in this thesis. In the Appendix section we have followed the unzipping of promoter DNA sequence using Optical Tweezers in an attempt to follow the temporal fluctuations occurring in biological reactions in real time and at a single molecule level.

The DNA binding proteins under starvation (Dps) from M. smegmatis are cage-like structures which internalize iron and bind DNA. They provide resistance to the cells from free radical damage, and physically protect the DNA from the harmful effects of reactive oxygen species by DNA compaction. The work compiled in this thesis has been an effort to study oligomerization and dynamics of iron metabolism by these nano-protein compartments. Chapter 1 gives a general introduction on stress, especially oxidative stress, and the ways bacteria fight back the host resistance systems. This has been elaborated from the point of view of the Dps proteins which is the focus of our work. Also, the competition for iron among the host and pathogens, and the modes of iron trafficking of the pathogens from host organisms has been summarized. Finally, the structural aspects of ferritin family proteins to which Dps belongs, has been discussed. Chapter 2 elaborates on the oligomerization pathways of the first M. smegmatis Dps MsDps1, which exists in vitro as two oligomeric forms. The GFP-tagging has been used to locate the Dps1 proteins by live cell imaging and the over-expression of these proteins during nutrient limiting conditions has been studied. The crystal structure of a point mutant F47E in the background of MsDps1, which shows no dodecamerization in vitro, has been solved. The possible ways of dodecamerization of MsDps1 has been concluded by analyzing the intermediates via glutaraldehyde cross-linking and native electrospray mass spectrometry. Chapter 3 documents the gating machinery of iron in MsDps2 protein, the second M. smegmatis Dps protein. Through graph theoretical approaches, a tight histidine-aspartate cluster was identified at the ferritin-like trimeric pore which harbors the channel for the entry and exit of iron. Sitespecific variants of MsDps2 were generated to disrupt this ionic knot, and the mutants were further assayed for ferroxidation, iron uptake and iron release properties. Our studies in MsDps2 show the importance of counter-acting positive and negatively charged residues for efficient assimilation and dispersion of iron. Chapter 4 describes crystallization studies of MsDps2 pore variants, done in an attempt to connect the changes in functional properties described in chapter 3, with structural alterations of the point mutants. We show here that the gating mechanism happens by alterations in side chain configuration at the pore and does not alter the over-all stability of the proteins. Chapter 5 is the final section where we have employed site specific mutations and cocrystallization studies to elucidate the behaviour of MsDps2 proteins upon the addition of iron. By studying the effect of substitutions at conserved sites near ferroxidation center, we attempt to arrive at a pathway which iron atoms take to reach the ferroxidation site. Also, by crystallization of proteins loaded with varying amounts of iron we tried to map the changes in the protein structure in the presence of its ligand. Chapter 6 concludes briefly the work that has been documented in this thesis. Appendix I relates the role of N-terminal tail for DNA binding in MsDp2. Appendix II gives the technical details of a modified protein preparation and oligomerization process for his-tagged MsDps1 protein. Appendix III gives the maps of the plasmids used in this study.

Plasmodium falciparum est un parasite protozoaire responsable de la forme la plus sévère de la malaria. Depuis quelques années, les cas de résistance aux antipaludiques sont devenus de plus en plus fréquents et de plus en plus répandus. En plus de sa résistance aux drogues actuellement disponibles, ce parasite reste jusqu' à aujourd'hui réfractaire aux vaccinations. L’identification de nouvelles approches basées sur l`inhibition spécifique de certaines de ses cibles moléculaires vitales est devenue une nécessité. La recombinase à site spécifique de P. falciparum (Pf-Int) est un enzyme qui a été récemment identifié dans le laboratoire à partir de PlasmoDB. Cette recombinase à site spécifique joue potentiellement un rôle clé dans le système de recombinaison nécessaire à la viabilité du parasite. Cette protéine de 490 acides aminés, soit ~57 kDa, contient une région C-terminale qui porte les résidus conservés du site catalytique des recombinases à tyrosine R-H-K-R-(H/W)-Y. La prédiction montre une région N-terminale qui ressemble à celle de l’intégrase du phage lambda avec un mélange de structures secondaires α et β.Lors de ces travaux, nous avons d’abord montré par RT-PCR que le gène (MAL13P1.42) qui code pour PF-Int est transcrit pendant le cycle intra-érythrocytaire avec un maximum pendant la phase schizont. Nous avons ensuite essayé de montrer l`implication de Pf-Int dans le cycle parasitaire. Ceci a été réalisé grâce à un parasite (KO: knock-out) dont le gène Pf-Int a été invalidé. Ces analyses montrent que Pf-Int n'a aucun impact apparent sur le cycle de développement intra-érythrocytaire du parasite, en particulier sur la durée du cycle et le taux de croissance. Au niveau moléculaire, nous avons également procédé à la production d'anticorps anti-Pf-Int en utilisant le fragment C-162 (Résidus 162-490). La comparaison des profils de marquage, par cet anticorps, des extraits protéiques du KO et du parasite sauvage par la technique de Western blot n'a pas permis d'identifier la protéine endogène dans le parasite sauvage. Dans le but de déterminer la localisation sub-cellulaire de Pf-Int, nous avons réalisé des essais de sur-expression de différentes protéines de fusion dans le parasite. Nous avons essayé de déterminer l’impact de trois codons d’initiation différents ainsi que l’impact de la présence de la région N-terminale (1-190aa) de Pf-Int sur sa localisation subcellulaire en utilisant une chimère entre la partie N-terminale et la protéine GFP. Lors de ces travaux, nous avons réussi à sur-exprimer différentes régions de Pf-Int sous forme recombinante dans E. coli. Nous l’avons d’abord caractérisé par des études biophysiques. Ainsi nous avons pu déterminer, par dichroïsme circulaire (CD), le contenu en structures secondaires de Pf-Int, qui est proche de celui des autres membres de la même famille. Nous avons également démontré sa stabilité par CD couplé à la dénaturation thermique. Le spectre RMN-1D a aussi pu être enregistré. La troisième partie de nos travaux a concerné l’identification des cibles ADN de Pf-Int. Deux stratégies de recherche de cibles par affinité ont été utilisées au laboratoire en utilisant une première bibliothèque de séquences synthétisées chimiquement et une deuxième bibliothèque formée de fragments d’ADN génomique de P. falciparum. Ces deux approches ont permis l’identification de deux séries de cibles ADN. Grace aux cibles ADN identifiées, nous avons pu démontrer l’interaction de différents fragments de Pf-Int avec ces cibles par des expériences de retard sur gel natif (EMSA). Nous avons aussi pu démontrer que les protéines recombinantes sont actives in vitro. En effet, ces dernières sont capables de former des complexes covalents en présence de l’ADN cible. La conservation de la protéine, ainsi que son expression différentielle nous laisse à penser que son rôle est certes loin d’être élucidé, mais que Pf-Int reste une cible potentielle pour P. falciparum. / Plasmodium falciparum is a protozoan parasite responsible for the most severe form of malaria. In recent years, cases of resistance to antimalarial drugs have become increasingly frequent and common. In addition to its resistance to drugs currently available, there is no vaccine available against this parasite till now. The identification of new approaches based on the specific inhibition of some of its molecular targets has become vital.The identification of the Pf-Int site specific recombinase in Plasmodium falciparum by analysis of PlasmoDB is a new opportunity to study the role of genetic variation in this parasite as it needs to adapt to its hosts. This ~ 57 kDa protein contains a C-terminal domain carrying the putative tyrosine recombinase conserved active site residues R-H-K-R-(H/W)-Y, an N-terminus with a predicted alpha-helical bundle and a mixed alpha-beta domain resembling Lambda-Int. Here, we show that the sequence is highly conserved among members of the Plasmodia. It is expressed differentially during distinct life stages as estimated by RT-PCR, namely with a peak in the schizont phase. We then tried to show the involvement of Pf-Int in the parasitic cycle. We were able to create a parasite where the Pf-Int gene was knocked-out. The comparison test showed that Pf-Int has apparently no impact on the intraerythrocytic developmental cycle of the parasite, particularly in the cycle length and the growth rate.At the molecular level, we produced two sets of anti-Pf-Int antibodies using the purified recombinant fragment C-162 (residues 162-490). Comparison of protein extracts from KO and wild parasite by Western blot technique using our antibody has failed to identify the endogenous protein in the wild type parasite.We also tried to determine the subcellular localization of Pf-Int and the role of possible alternate initiation codons by over-expressing different constructs in the parasite Plasmodium falciparum. In order to determine the impact of the N-terminal region (1-190aa) of Pf-Int on its subcellular localization, we also created a chimeric protein using a fusion of Pf-Int(1-190aa) with the GFP. We successfully expressed a variety of the recombinant form of Pf-Int in E. coli. We have first determined its secondary structure content by circular dichroism (CD) and its solution stability by thermal denaturation-CD. An 1-D NMR spectrum was also recorded. The third part of our work has involved the identification of the DNA targets of Pf-Int. Two search strategies conducted in the laboratory using a library of chemically synthesized sequences and a second library made of fragments of genomic DNA of P. falciparum. Both approaches have allowed the identification of two sets of target DNA. Secondly, electrophoretic mobility shift assays (EMSA) were used to show its affinity and specificity for DNA. The recombinant proteins were shown to be functional as they form a covalent complex with DNA. Thus Pf-Int could be a potential agent that binds to and alters DNA, either in a specific or in random fashion. Its conservation and differential expression leads us to conclude that although its role is far from being understood, Pf-Int remains a key target for P. falciparum.