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1
Takahshi, Naoko. "Flagella synthesis in Rhodobacter sphaeroides WS8." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259820.
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Koripella, Srihari Nagendra Ravi Kiran. "Characterizing Elongation of Protein Synthesis and Fusidic Acid Resistance in Bacteria." Doctoral thesis, Uppsala universitet, Institutionen för cell- och molekylärbiologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-207924.
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Protein synthesis is a highly complex process executed by the ribosome in coordination with mRNA, tRNAs and translational protein factors. Several antibiotics are known to inhibit bacterial protein synthesis by either targeting the ribosome or the proteins factors involved in translation. Fusidic acid (FA) is a bacteriostatic antibiotic that blocks polypeptide chain elongation by locking elongation factor-G (EF-G) on the ribosome. Mutations in fusA, the gene encoding bacterial EF-G, confer high-level of resistance towards FA. Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by acquiring secondary mutations. In order to understand the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that, the causes for fitness loss in the FA-resistant mutant F88L are resulting from significantly slower tRNA translocation and ribosome recycling. Analysis of the crystal structures, together with the results from our biochemical studies enabled us to propose that FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome. EF-G is a G-protein belonging to the GTPase super-family. In all the translational GTPases, a conserved histidine (H92 in E. coli EF-G) residue, located at the apex of switch II in the G-domain is believed to play a crucial role in ribosome-stimulated GTP hydrolysis and inorganic phosphate (Pi) release. Mutagenesis of H92 to alanine (A) and glutamic acid (E) showed different degree of defect in different steps of translation. Compared to wild type (WT) EF-G, mutant H92A showed a 10 fold defect in ribosome mediated GTP hydrolysis whereas the other mutant H92E showed a 100 fold defect. However, both the mutants are equally defective in single round Pi release (100 times slower than WT). When checked for their activity in mRNA translocation, H92A and H92E were 10 times and 100 times slower than WT respectively. Results from our tripeptide formation experiments revealed a 1000 fold defect for both mutants. Altogether, our results indicate that GTP hydrolysis occurs before tRNA translocation, whereas Pi release occurs probably after or independent of the translocation step. Further, our results confirm that, His92 has a vital role residue in ribosome-stimulated GTP hydrolysis and Pi release.
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Fowler, Colleen Marie. "Evaluation of 2-Hydroxy-4-(methylthio) Butanoic Acid Isopropyl Ester and Methionine Supplementation on Efficiency of Microbial Protein Synthesis and Rumen Bacterial Populations." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1248875016.
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Haas, R. Matthew. "Synthesis and characterization of phosphono-CheY from Thermotoga maritima /." Electronic version (PDF), 2007. http://dl.uncw.edu/etd/2007-1/haasr/rmatthewhaas.html.
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Lovmar, Martin. "Macrolide Antibiotics in Bacterial Protein Synthesis." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6009.
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Zhang, Guangtao. "Design, synthesis, and evaluation of cholera toxin inhibitors and [alpha]-helix mimetics of dormancy survival regulator /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/8485.
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Johansson, Magnus. "Rate and Accuracy of Bacterial Protein Synthesis." Doctoral thesis, Uppsala universitet, Struktur- och molekylärbiologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-171040.
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High levels of accuracy in transcription, aminoacylation of tRNA, and mRNA translation are essential for all life forms. However, high accuracy also necessarily means large energy dissipation and slow kinetics. Therefore, in vivo there is a fine tuned balance between rate and accuracy of key chemical reactions. We have shown that in our optimized in vitro bacterial protein synthesis system we have in vivo compatible rate and accuracy of ribosomal protein elongation. Our measurements of the temperature and the pH dependence of peptide bond formation with native substrates also suggest that the chemical step of peptidyl transfer, rather than tRNA accommodation, limits the rate of peptide bond formation. This work has made it possible to study ribosomal peptidyl transfer with native substrates. Furthermore, we have developed a general theoretical model for the rate-accuracy trade-off in enzymatic reactions. When considering this trade-off for protein synthesis in the context of the living bacterial cell, where cognate aa-tRNAs compete for ribosome binding with an excess of non-cognate aa-tRNAs, the model predicts an accuracy optimum where the inhibitory effect of non-cognate substrate binding and the efficiency loss due to high discard rate of cognate aa-tRNAs are minimized. However, these results also show that commonly used biochemical systems for protein synthesis studies operate at exceptionally suboptimal conditions. This makes it difficult, if not impossible, to relate the biochemical data to protein synthesis in the living cell. To validate our theoretical model we developed a method, based on variation of the concentration of Mg2+ ions in the buffer, to study the rate-accuracy trade-off of bacterial protein synthesis in vitro. We found a linear trade-off between rate and accuracy of tRNA selection on the ribosome, from which we could estimate the maximal accuracy. Exploiting this method for a complete set of single-mismatch readings by one tRNA species, we found simple patterns of genetic code reading, where the accuracy was highest for the second and lowest for the third codon position. The results bridge the gap between in vivo and in vitro protein synthesis and allow calibration of our test tube conditions to those of the living cell.
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Zhang, Jingji. "Accuracy of mRNA Translation in Bacterial Protein Synthesis." Doctoral thesis, Uppsala universitet, Struktur- och molekylärbiologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-262901.
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Reading of messenger RNA (mRNA) by aminoacyl-tRNAs (aa-tRNAs) on the ribosomes in the bacterial cell occurs with high accuracy. It follows from the physical chemistry of enzymatic reactions that there must be a trade-off between rate and accuracy of initial tRNA selection in protein synthesis: when the current accuracy, the A-value, approaches its maximal possible value, the d-value, the kinetic efficiency of the reaction approaches zero. We have used an in vitro system for mRNA translation with purified E. coli components to estimate the d- and A-values by which aa-tRNAs discriminate between their cognate and near cognate codons displayed in the ribosomal A site. In the case of tRNALys, we verified the prediction of a linear trade-off between kinetic efficiency of cognate codon reading and the accuracy of codon selection. These experiments have been extended to a larger set of tRNAs, including tRNAPhe, tRNAGlu, tRNAHis, tRNACys, tRNAAsp and tRNATyr, and linear efficiency-accuracy trade-off was observed in all cases. Similar to tRNALys, tRNAPhe discriminated with higher accuracy against a particular mismatch in the second than in the first codon position. Remarkably high d-values were observed for tRNAGlu discrimination against a C-C mismatch in the first codon position (70 000) and for tRNAPhe discrimination against an A-G mismatch in the second codon position (79 000). At the same time, we have found a remarkably small d-value (200) for tRNAGlu misreading G in the middle position of the codon (U-G mismatch). Aminoglycoside antibiotics induce large codon reading errors by tRNAs. We have studied the mechanism of aminoglycoside action and found that the drug stabilized aminoacyl-tRNA in a codon selective in relation to a codon non-selective state. This greatly enhanced the probability of near cognate aminoacyl-tRNAs to successfully transcend the initial selection step of the translating ribosome. We showed that Mg2+ ions, in contrast, favour codon non-selective states and thus induce errors in a principally different way than aminoglycosides. We also designed experiments to estimate the overall accuracy of peptide bond formation with, including initial selection accuracy and proofreading of tRNAs after GTP hydrolysis on EF-Tu. Our experiments have now made it possible to calibrate the accuracy of tRNA selection in the test tube to that in the living cells. We will now also be able to investigate the degree to which the accuracy of tRNA selection has been optimized for maximal fitness.
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Nagalingam, Anil. "Towards the total synthesis of bacterial immunity proteins." Thesis, University of Leeds, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.427784.
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Shanley, Samantha Jane. "A glycopore for bacterial sensing." Thesis, University of Oxford, 2009. http://ora.ox.ac.uk/objects/uuid:06fe9bce-6bf2-4f61-b4d8-014cb9df3fc0.
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Increasing antibiotic resistance has created a need to develop rapid and reliable methods to identify bacteria and provide pertinent information to ensure suitable antibiotics or sugar therapeutics can be chosen for treatment. Carbohydrate structures attached to proteins on host cell surfaces provide a binding point for many pathogens, including bacteria. These structures can be mimicked using single monosaccharides glycosylated to alpha-hemolysin (alpha-HL). Alpha-HL is a beta-barrel pore-forming toxin secreted by Staphylococcus aureus that forms an SDS stable heptamer, which can be expressed by coupled in vitro transcription and translation and purified by polyacrylamide gel electrophoresis. The purified heptamers can be reconstituted into planar lipid bilayers and studied at the single channel level. Through single channel recordings the effects of sugar-linker lengths, different glycans and the interaction between the ‘Glycopore’ and sugar binding molecules can be studied. The glycopore, therefore, acts as a scaffold for analysing protein-sugar interactions. Studies in this thesis have focused on the synthesis of carbohydrates for site-selective protein glycosylation; cloning and in vitro transcription translation of alpha-HL monomers; and glycosylation and oligomerisation of alpha-HL to form glycopores suitable for lectin-binding studies. Lectins DC-SIGN and FimH have been expressed in Escherichia coli and these lectins as well as others have been screened using alpha-HL glycopores. The glycopores have also been investigated with bacteria in serum in a controlled molecule-specific manner using single-channel electrical recording. In this work glycosylated alpha-HL-monomers have been found to form stable heptamers which can be formed by oligomerisation on red blood cell membranes. The purified glycopores were reconstituted into planar lipid bilayers and studied at the single-channel level. Through single-channel recordings an optimised glycopore has been shown to be effective in distinguishing lectins alone and in a mixture and has afforded qualitative and quantitative information about the binding interactions between carbohydrates and sugar binding proteins. Furthermore, the glycopore has been used to sense bacteria which may provide an insight into modes of bacterial infection. In addition, a multivalent glycopore has been formed which has proved preliminary information about the effects of multivalency in lectin binding. The design and synthesis of non-beta-lactam antibiotic candidates and their evaluation has also been carried out.
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Swallow, Isabella Diane. "Probes for bacterial ion channels." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:d42d13dd-dd0c-451b-bd00-e06f84350335.
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Using three complementary approaches, this work sought to tackle the widespread problem of antibiotic resistance. To circumvent the resistance mechanisms developed by bacteria, it is necessary to establish drug candidates that act on novel therapeutic targets, such as the ion channels used by bacteria to modulate homeostasis. Examples include the potassium efflux channel, Kef, and the mechanosensitive channel of small conductance, MscS, which are not found in humans. How these targets function must be well understood before drug candidates can be developed, as such, their identification and investigation is often accompanied by the evolution of the analytical techniques used to study them. Membrane protein mass spectrometry is one technique showing potential in the study of ion channels. However, spectra can be clouded by the detergents used to solubilise ion channels from their native membranes. Undertaken herein was the synthesis of some fluorescent glycolipid detergents, which it was hypothesised could be encouraged to dissociate from ion channels via laser-induced excitation within the gas phase of a mass spectrometer, thereby improving the clarity with which spectra can be obtained. For Kef, an unconfirmed mechanism of action had previously been proposed. To explore the suggestion that sterically-demanding central residues are important for channel activation, solid phase peptide synthesis was used to isolate three tripeptide analogues of N-ethylsuccinimido glutathione, a known activator with a high affinity for Kef. A competition fluorescence assay suggested these tripeptides bound to Kef with an affinity lower than predicted, allowing the conclusion that a more detailed assessment of the steric bulk required for activation was necessary before a mechanism of action could be confirmed. Lysophosphatidylcholine has been shown to activate MscS, although it is not known how. Affinity chromatography between MscS and lysophosphatidylcholine was proposed as a means by which specific binding interactions could be investigated. For this technique an amino-derivative of lysophosphatidylcholine was necessary and its challenging synthesis is also detailed herein.
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Hjerdt-Goscinski, Gunilla. "Antibiotic-induced Bacterial Toxin Release – Inhibition by Protein Synthesis Inhibitors." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4692.
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Markovski, Monica. "Bacterial Cell Wall Synthases Require Outer Membrane Lipoprotein Cofactors." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10146.
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To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton. The PG synthases that build this structure are called penicillin-binding proteins (PBPs). Since they are the targets of penicillin and related antibiotics, the structures and in vitro biochemical functions of the PBPs have been extensively studied. However, the in vivo functions of the PBPs and the factors they work with to build the PG meshwork remain poorly understood. PBPs work in the context of multicomponent complexes organized by cytoskeletal elements. A major outstanding question has been whether or not these complexes contain factors required for PBP function. I addressed this using Escherichia coli as a model system by taking advantage of the synthetic lethal phenotype resulting from simultaneous inactivation of the major PG synthases: PBP1a and PBP1b. Using a screen for mutants synthetically lethal with the inactivation of PBP1b, I identified LpoA as a factor required for PBP1a function. A colleague in the lab performed the analogous screen for mutants synthetically lethal with the inactivation of PBP1a and identified LpoB as a factor required for PBP1b function. We showed that the Lpo factors are outer membrane lipoproteins that form specific trans-envelope complexes with their cognate PBPs in the inner membrane and that LpoB can stimulate the activity of PBP1b in vitro. Our results reveal unexpected complexity in the control of PBP activity and indicate that they likely receive regulatory input from the outer membrane in addition to cytoskeletal elements in the cytoplasm. To investigate the role of LpoB in morphogenesis further, I took a genetic approach that has identified PBP1b* variants capable of functioning in vivo in the absence of LpoB. Preliminary characterization of these variants indicates that LpoB has cellular functions in addition to PBP1b activation and that LpoB may be important for coordinating the two different catalytic activities of PBP1b. Future study of these mutants is likely to uncover important insights into PBP function and their control by the Lpo factors. These insights may open new avenues for the development of novel therapeutics that target the PBPs.
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Zhao, Yue. "Synthetic probes for bacterial lipids and dimerizing proteins." Thesis, Boston College, 2015. http://hdl.handle.net/2345/bc-ir:104623.
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Thesis advisor: Eranthie WeerapanaThis thesis includes two projects: “Bacteria-selective borono-peptides” and “A split ligand for lanthanide binding: facile evaluation of dimerizing proteins”. In both projects, de novo designed molecules were synthesized, optimized and incorporated into peptides. These synthetic molecular tools allow selective targeting of bacterial cell membranes and analyzing the dynamic associations of membrane-embedded proteins. 1. Bacteria-selective borono-peptides As the antibiotic resistance continues to grow, bacterial infection becomes one of the major threats to global public health. Currently, almost all the bacteria targeting strategies employ non-covalent driving forces, including charge-charge interactions, hydrophobic interactions and the formation of hydrogen bonds, to achieve bacterial selectivity. Towards novel bacteria targeting molecules, we have recruited reversible covalent chemistry in the development of bacteria-selective peptides. Targeting the diol-rich environment of a bacterial surface, we have designed and synthesized several unnatural amino acids that contain boronic acid moieties. Taking advantage of the boronic acid-diol reaction and multivalency effect, our borono-peptides are found to selectively recognize bacteria over mammalian cells. The sensitivity of the binding event to carbohydrate competitors gives a safe and facile approach to regulate molecular association with bacterial cells. This design may find applications in the fields of bacterial detection, imaging and antimicrobial drug delivery. 2. A split ligand for lanthanide binding: facile evaluation of dimerizing proteins Protein dimerization is a ubiquitous phenomenon in biology and plays a critical role in transcription regulations and various signaling processes. Methods that allow facile detection and quantification of protein dimers are highly desirable for evaluating protein dimerization in physiology and disease. Meanwhile, luminescence of lanthanides is attractive for biological applications due to its long lifetime and sharp emission profiles. We have developed a split lanthanide binding ligand that allows facile evaluation of dimerizing proteins. The fast lanthanide–ligand (dis)association allows us to monitor the dynamic behavior of dimerizing proteins. We have demonstrated the successful application of our assay on both soluble and transmembrane proteins in complex biological milieu. The split lanthanide ligand is cysteine reactive, and therefore should be readily applicable to a variety of proteins of interest
Thesis (PhD) — Boston College, 2015
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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SANCHEZ-LOPEZ, ROSANA. "Partie i : etude des mecanismes impliques dans la resistance au sel chez une bacterie halophile- partie ii : etude des relations structure-fonction de metalloproteinases de la famille collagenase." Université Louis Pasteur (Strasbourg) (1971-2008), 1989. http://www.theses.fr/1989STR13020.
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These en deux parties: d'une part etude d'une bacterie en milieu salin. L'addition de proline, choline, permet la croissance de la bacterie. Analyse de la composition proteique de la membrane bacterienne. D'autre part, etude de metalloproteinases et de certaines sequences impliquees dans le site actif de l'enzyme et dans l'activation du zymogene
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Shipley, Paul R. "The biosynthesis of the thiopeptide antibiotic thiostrepton /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/11559.
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Wang, Wenjian. "Developing functional peptides as synthetic receptors, binders of protein and probes for bacteria detection:." Thesis, Boston College, 2021. http://hdl.handle.net/2345/bc-ir:109036.
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Thesis advisor: Jianmin GaoThesis advisor: Eranthie Weerapana
Nature has developed a generous number of peptides carrying out various essential functions in all living organisms. Human body produces peptides as signaling molecules, such as hormones, to transmit messages from cell to cell and regulate metabolic homeostasis. Microbes synthesize peptides as antibiotics to inhibit the growth of other microorganisms. These peptides display an exceeding diversity of amino acid composition, peptide sequence, secondary structure and post-translational modification. Inspired by nature, researchers have developed peptides as a unique modality of therapeutics, combining the best attributes of small-molecule drugs and protein-based biopharmaceuticals. This work has sought to explore the potential of peptides as synthetic receptors, binders of protein and probes for bacteria detection. The research started from a foldable cyclic peptide scaffold, prolinomycin, a proline-rich analogue of valinomycin. The peptide can chelate a potassium ion folding into a drum like structure, which provides a platform to display and preoganize functional side chains for target binding. We first investigated its folding behavior under physiological conditions. We demonstrate that the metal-assisted folding of the prolinomycin scaffold tolerates various side chain mutations. The stability of the structure can be improved by introducing crosslinking moieties. Based on this scaffold, we rationally designed synthetic receptors of various amines by utilizing iminoboronate chemistry with acetylphenyl boronic acid (APBA). Furthermore, I pursued phage display, a powerful technique to develop high affinity peptide binders of protein targets. Proteins are the most appealing targets for drug development and disease biomarkers discovery. We chose sortase A (SrtA) as a model target protein to screen for potent peptide binders. A peptide inhibitor of sortase A with single-digit micromolar affinity was identified from a cyclic peptide library displayed by phage. In addition, from the chemically modified phage display peptide library presenting APBA motifs, peptide binders with specificity and micromolar affinity towards SrtA were discovered. Instead of binding to the active site, the peptide could recognize the surface of the protein. Additionally, to further expand the chemical space of phage display, I constructed a phage display peptide library presenting N-terminal cysteine (NCys) which can undergo site-specific chemical modifications. Two pieces of chemistry were applied, including thiazolidino boronate (Tzb) mediated acylation reaction of NCys and 2-cyanobenzothiazole (CBT)-NCys condensation. The site-specific dual modifications on NCys and internal Cys of phage-encoded peptides were achieved. Furthermore, a strategy to N, S-doubly label NCys via an alternative pathway of CBT condensation was reported, which presents a significant addition to the toolbox for site-specific protein modifications. Finally, by functionalizing graphene field effect transistors (G-FET) with peptide probes, we developed the first selective, electrical detection of the pathogenic bacterial species Staphylococcus aureus and antibiotic resistant Acinetobacter baumannii on a single platform. Overall, peptides provide enormous opportunities for therapeutics development. Research herein demonstrated principles of peptide design for specific molecular recognition. Novel chemistry strategies have been developed to expand the molecular diversity of peptide libraries. We believed that the advances in peptide design and screening would promote peptide-based drug discovery
Thesis (PhD) — Boston College, 2021
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Tobin, Christina. "Removal and Replacement of Ribosomal Proteins : Effects on Bacterial Fitness and Ribosome Function." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-150401.
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Protein synthesis is a complex process performed by sophisticated cellular particles known as ribosomes. Although RNA constitutes the major structural and functional component, ribosomes from all kingdoms contain an extensive array of proteins with largely undefined functional roles. The work presented in this thesis addresses ribosomal complexity using mutants of Salmonella typhimurium to examine the physiological effects of ribosomal protein (r-protein) removal and orthologous replacement on bacterial fitness and ribosome function. The results of paper I demonstrate that removal of small subunit protein S20 conferred two independent translation initiation defects: (i) a significant reduction in the rate and extent of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in subunit association. The topographical location of S20 in mature 30S subunits suggests that these perturbations are the result of improper orientation of helix 44 of the 16S rRNA when S20 is absent. In paper II we show that the major functional impairment associated with loss of large subunit protein L1 manifested as an increase in free ribosomal subunits at the expense of translationally active 70S particles. Furthermore, the formation of free ribosomal subunits was imbalanced suggesting that L1 is required to suppress degradation or promote formation of 30S subunits. Compensatory evolution revealed that mutations in other large subunit proteins mitigate the cost of L1 removal, in one case seemingly via an increase in 70S complex formation. As shown in paper III, the large fitness costs associated with complete removal of r-proteins is in contrast to the generally mild costs of orthologous protein replacement, even in the absence of a high degree of homology to the native protein. This clearly demonstrates the robustness and plasticity of the ribosome and protein synthesis in general and it also implies that functional constraints are highly conserved between these proteins. The findings of paper III also allowed us to examine the barriers that constrain horizontal gene transfer and we find that increased gene dosage of the sub-optimal heterologous protein may be an initial response to stabilize deleterious transfer events. Overall the results highlight the requirement of r-proteins for the maintenance of ribosomal structural integrity.
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Ieong, Ka-Weng. "Rate and Accuracy of Bacterial Protein Synthesis with Natural and Unnatural Amino Acids." Doctoral thesis, Uppsala universitet, Struktur- och molekylärbiologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-235534.
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This thesis addresses different questions regarding the rate, efficiency, and accuracy of peptide bond formation with natural as well as unnatural amino acids: Which step is rate-limiting during peptide bond formation? How does the accuracy vary with different transfer RNAs (tRNAs) and codons and how is it relevant to the living cells? Does proofreading selection of codon reading occur in a single- or multi-step manner as theoretically suggested? How does the E. coli translation system discriminate unnatural amino acids? Based on that, how to improve the incorporation efficiencies of unnatural amino acids? Based on the study on pH dependence of peptide bond formation, we show that the rate of the chemistry of peptidyl transfer to aminoacyl-tRNA (AA-tRNA) Gly-tRNAGly or Pro-tRNAPro limits the rate of peptide bond formation at physiological pH 7.5, and this could possibly be true for peptidyl transfer to all natural AA-tRNAs at physiological condition. By studying the efficiency-accuracy trade-off for codon reading by seven AA-tRNA containing ternary complexes, we observe a large variation on the accuracy of initial codon selection and identify several error hot-spots. The maximal accuracy varied 400-fold from 200 to 84000 depending on the tRNA identity, the type and position of the mismatches. We also propose a proofreading mechanism that contains two irreversible steps in sequence. This could be highly relevant to the living cells in relation to maintaining both high accuracy and high efficiency in protein synthesis. Finally, we show that peptide bond formation with small and large non-N-alkylated L- unnatural amino acids proceed at rates similar to those with natural amino acids Phe and Ala on the ribosome. Interestingly, the large side chain of the bulky unnatural amino acid only weakens its binding for elongation factor Tu (EF-Tu) but not slows down peptidyl transfer on the ribosome. Our results also suggest that the efficiency of unnatural amino acid incorporation could be improved in general by increasing EF-Tu concentration, lowering the reaction temperature and / or using tRNA bodies with optimal affinities for EF-Tu in the translation system.
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Blades, Gareth. "Re-engineering bacterial two-component signalling systems." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:2865c02d-c208-45fa-8108-d8ced9486c19.
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Bacteria use Two Component Systems (TCS) to sense and respond to changes in their external environment. TCS are used to navigate to nutrients or away from toxins (chemotaxis) and to adapt to changes in osmolarity (osomosensing). TCS are composed of a histidine protein kinase (HPK) which trans-autophosphorylates in response to environmental change, transferring the phosphoryl group to a cognate response regulator (RR). Phosphorylated RRs modulate an output response such as protein-protein interaction for chemotaxis, and transcription for osmosensing. RRs are composed of a conserved amino terminal REC domain, and where present a variable effector domain. CheY, the chemotaxis RR, contains only a REC domain, whilst OmpR, the osmosensing RR, also contains a DNA binding effector domain. Recently, TCS have been used in synthetic biology applications due to their modularity and conserved signalling mechanism. This thesis aimed to investigate whether it was possible to design a synthetic TCS composed of fused chemotaxis and osmosensing components. Synthetic RRs were designed, fusing the highly conserved REC domains of CheY and OmpR upstream of the OmpR effector domain. REC domains were fused across the α4-β5-α5 region, a region which transmits REC domain phosphorylation into effector domain activation. Synthetic RRs were designed to undergo phosphotransfer to their fused REC domains from the chemotaxis HPK, CheA, activate the attached OmpR effector domain and bind promoter DNA. Four chimeric RRs were created, although only three were structurally viable; F2, F3 and F4. Each fusion bound CheA, and F3 and F4 bound CheA with a significantly higher affinity than CheY. The chimeric RRs could all be phosphorylated byCheA-P; F4 and F3 were phosphorylated to wild-type levels. DNA binding affinitywas investigated with fluorescence anisotropy, hosphorylated and unphosphorylated F3 could not bind promoter DNA. F2 bound promoter DNA regardless of phosphorylation state. These data indicate that phosphorylation of the F2 REC domain does not lead to activation of the effector domain. F2 is likely to be constitutively active suggesting a previously unknown role for OmpR α5 as a mediator of effector domain activation. Furthermore, using a simple fusion approach to design RRs is not a viable method to create a synthetic TCS with a controllable output.
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Borg, Anneli. "Mechanisms and Inhibition of EF-G-dependent Translocation and Recycling of the Bacterial Ribosome." Doctoral thesis, Uppsala universitet, Struktur- och molekylärbiologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-258990.
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The GTPase elongation factor G (EF-G) is an important player in the complex process of protein synthesis by bacterial ribosomes. Although extensively studied much remains to be learned about this fascinating protein. In the elongation phase, after incorporation of each amino acid into the growing peptide chain, EF-G translocates the ribosome along the mRNA template. In the recycling phase, when the synthesis of a protein has been completed, EF-G, together with ribosome recycling factor (RRF), splits the ribosome into its subunits. We developed the first in vitro assay for measuring the average time of a complete translocation step at any position along the mRNA. Inside the open reading frame, at saturating EF-G concentration and low magnesium ion concentration, translocation rates were fast and compatible with elongation rates observed in vivo. We also determined the complete kinetic mechanism for EF-G- and RRF-dependent splitting of the post-termination ribosome. We showed that splitting occurs only when RRF binds before EF-G and that the rate and GTP consumption of the reaction varies greatly with the factor concentrations. The antibiotic fusidic acid (FA) inhibits bacterial protein synthesis by binding to EF-G when the factor is ribosome bound, during translocation and ribosome recycling. We developed experimental methods and a theoretical framework for analyzing the effect of tight-binding inhibitors like FA on protein synthesis. We found that FA targets three different states during each elongation cycle and that it binds to EF-G on the post-termination ribosome both in the presence and absence of RRF. The stalling time of an FA-inhibited ribosome is about hundred-fold longer than the time of an uninhibited elongation cycle and therefore each binding event has a large impact on the protein synthesis rate and may induce queuing of ribosomes on the mRNA. Although ribosomes in the elongation and the recycling phases are targeted with similar efficiency, we showed that the main effect of FA in vivo is on elongation. Our results may serve as a basis for modelling of EF-G function and FA inhibition inside the living cell and for structure determination of mechanistically important intermediate states in translocation and ribosome recycling.
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Mačvanin, Mirjana. "The physiological cost of antibiotic resistance /." Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3761.
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Nguyen, Fabian [Verfasser], and Daniel [Akademischer Betreuer] Wilson. "Antibiotics inhibiting bacterial protein synthesis, and novel resistance mechanisms / Fabian Nguyen ; Betreuer: Daniel Wilson." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2018. http://d-nb.info/1162840595/34.
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Huang, Hexian. "Regulations of export and chain length of extracellular bacterial polysaccharides." Thesis, University of St Andrews, 2013. http://hdl.handle.net/10023/4441.
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Many Gram-positive and Gram-negative bacteria produce an additional thick layer of carbohydrate polymers on the cell wall surface. These capsules (capsular polysaccharides; CPS) play critical roles in interactions between bacteria and their environments (Whitfield, 2006). This is especially important in infection processes since for both Gram-negative and Gram-positive pathogens CPS is the point of first contact with the host immune system (Whitfield, 2006). However, the details of CPS biosynthesis and assembly mechanisms are still unclear. Therefore, we embarked on structural and kinetic studies of the proteins Wzc, Wza and Wzb/ Cps4B from the Wzy-dependent pathway, as well as the protein WbdD from the ATP-binding cassette (ABC) transporter dependent system. Full-length Wzc failed to crystallise due to the presence of large disordered regions and the overall difficulty of membrane protein crystallisation. A truncated version of Wzc (1-480) without the C-terminal tyrosine kinase domain was crystallised and diffracted to 15 Å in house. A previous study suggested Wza and Wzc form a functional complex (Whitfield, 2006), so Wza was also studied. Since the full-length Wza structure is available (C. Dong et al., 2006), Pulsed electron–electron double resonance spectroscopy (PELDOR) was used to study the conformational change. The PELDOR spectroscopy distance fingerprint of Wza was determined. These data also confirmed that PELDOR is a powerful tool to study large, highly symmetrical membrane proteins and can be used to study other complex membrane protein systems, such as ion channels or transporters. The crystal structure of Wzb the cognate phosphatase of Wzc was determined to 2.2 Å. Also Cps4B, which is a functional homologue of Wzb but has a completely unrelated sequence, was crystallised in two crystal forms. Form I and II Cps4B crystals diffracted to 2.8 Å and 1.9 Å resolution in house, respectively. The full-length WbdD failed to crystallise due to the presence of large disordered regions. Therefore, a shorter construct, WbdD₅₅₆ (1-556) was cloned and crystallised. The structure was determined to 2.2 Å. WbdD is a bifunctional enzyme consisting of a methyltransferase (MTase) and a kinase domain. In order to better understand the function of this protein, a variety of techniques were used, such as the ADP-Glo kinase assay, Nuclear magnetic resonance (NMR) spectroscopy, small angle X-ray scattering (SAXS) and X-ray crystallography. The various findings in the current projects provide meaningful insights towards a better understanding of the CPS biosynthesis and assembly mechanisms, which may contribute to a more intensive study identifying inhibitors and beginning to unravel the mechanism of chain length regulation.
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Rajkovic, Andrei. "Promoting Bacterial Synthesis of Oligo-prolines by Modifying Elongation Factor P Post-translationally." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469123846.
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Cao, Mingle [Verfasser], and Peter [Akademischer Betreuer] Graumann. "Bio-synthesised LytC protein kills bacteria and the study of protein dynamics in B. subtilis / Mingle Cao. Betreuer: Peter Graumann." Marburg : Philipps-Universität Marburg, 2016. http://d-nb.info/1108765750/34.
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Karlsson, Katarina Flemmer. "Synthesis, conformational analysis, and biological evaluation of peptides from E. coli P pilus proteins." Lund : Organic Chemistry 2, Lund Institute of Technology, Lund University, 1997. http://catalog.hathitrust.org/api/volumes/oclc/39777038.html.
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Huang, Po-Yi. "Study on Bacterial Protein Synthesis System toward the Incorporation of D-Amino Acid & Synthesis of 2'-deoxy-3'-mercapto-tRNA." Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11365.
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Life is anti-entropic and highly organized phenomenon with two characteristics reinforcing each other: homochirality and the stereospecific catalysis of chemical reactions. The exclusive presence of L-amino acids and R-sugars in living world well depict this. Hypothetically, the amino acids and sugars of reverse chirality could form a parallel kingdom which is highly orthogonal to the present world. The components from this mirror kingdom, such as protein or nucleic acid, will be much more resistant to the defensive mechanism of present living system, which could be of great value. Therefore, by gradually rewiring the present bio-machineries, we look to build a bridge leading us to the space of mirror-imaged biomolecules. We begin by investigating protein synthesis with mirror amino acid since most amino acids contain one chiral center to be inversed comparing to sugars. In this work, we analyzed three stages critical for the incorporation of D-amino acid into ribosomal protein synthesis: amino acylation, EF-Tu binding of amino acyl-tRNA and delivery bias, and ribosome catalyzed peptidyl transfer. We have demonstrated that the affinity between EF-Tu and amino acyl-tRNA plays critical role on D-amino acid incorporation, and built a platform aimed to select for ribosome tolerating D-amino acid better.Chemistry and Chemical Biology
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Frazier, Ashley Denise. "An Investigation of Bacterial Ribonucleases as an Antibiotic Target." Digital Commons @ East Tennessee State University, 2012. https://dc.etsu.edu/etd/1417.
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Antibiotics have been commonly used in medical practice for over 40 years. However, the misuse and overuse of current antibiotics is thought to be the primary cause for the increase in antibiotic resistance.
Many current antibiotics target the bacterial ribosome. Antibiotics such as aminoglycosides and macrolides specifically target the 30S or 50S subunits to inhibit bacterial growth. During the assembly of the bacterial ribosome, ribosomal RNA of the 30S and 50S ribosomal subunits is processed by bacterial ribonucleases (RNases). RNases are also involved in the degradation and turnover of this RNA during times of stress, such as the presence of an antibiotic. This makes ribonucleases a potential target for novel antibiotics.
It was shown that Escherichia coli mutants that were deficient for RNase III, RNase E, RNase R, RNase G, or RNase PH had an increase in ribosomal subunit assembly defects. These mutant bacterial cells also displayed an increased sensitivity to neomycin and paromomycin antibiotics. My research has also shown that an inhibitor of RNases, vanadyl ribonucleoside complex, potentiated the effects of an aminoglycoside and a macrolide antibiotic in wild type Escherichia coli, methicillin sensitive Staphylococcus aureus, and methicillin resistant Staphylococcus aureus.
RNases are essential enzymes in both rRNA maturation and degradation. Based on this and previous work, the inhibition of specific RNases leads to an increased sensitivity to antibiotics. This work demonstrates that the inhibition of RNases might be a new target to combat antibiotic resistance.
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Daniels, Craig. "Characterisation of proteins involved in Shigella flexneri O-antigen biosynthesis." Title page, abstract and contents only, 1999. http://web4.library.adelaide.edu.au/theses/09PH/09phd186.pdf.
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Corrigenda pasted onto back end-papers. Bibliography: leaves 163-182. Analyses the proteins involved in Shigella flexneri O-antigen biosynthesis at the molecular level in order to gain a more concise understanding of the biosynthesis machinery and how it functions.
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Rani, Suriani Abdul. "Spatial Patterns of DNA Replication, Protein Synthesis, and Oxygen Concentration within Bacterial Biofilms Reveal active and Inactive Regions." Thesis, Montana State University, 2006. http://etd.lib.montana.edu/etd/2006/rani/RaniS0806.pdf.
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Biofilms harbor both active and inactive cells and it is a challenge to characterize the spatial and population heterogeneity of specific activities within a biofilm. Spatial patterns of DNA replication and protein synthetic activity were imaged by techniques developed using staphylococcal systems. The first technique measures DNA synthetic activity by pulse-labeling with the thymidine analog 5-bromo-2-deoxyuridine (BrdU) followed by immunofluorescent detection of brominated DNA. The second technique makes use of an inducible green fluorescent protein construct that can be used to detect the capacity for de novo protein synthesis. These techniques were applied to biofilms grown in three different reactor systems. In all cases, measurements revealed that even in simple single-species biofilms, complex spatial distributions of anabolic activity occur. In a colony biofilm system, two distinct regions of DNA synthetic activity were observed, one close to the nutrient interface and another adjacent to the air interface. A similar pattern was measured by GFP induction. The dimensions of DNA synthetic activity ranged from 25 to 31 µm and the average protein synthetic activity ranged from 36 to 38 µm at the air interface. When pure oxygen was introduced, a wider zone of active DNA replication (45 µm) and GFP synthesis (59 µm) was measured at the gas interface. Oxygen penetration calculated (26µm) corresponds with the zones of respiratory activity (19 to 38 µm), DNA synthetic activity and protein synthetic activity measured at the air interface. The dimensions of DNA synthetic activity and protein synthesis activity at the nutrient interface ranged from 13 µm to 19 µm. The addition of glucose to the media increased the zone of protein synthesis at the nutrient interface to 33 µm. Stratified patterns of activity were also observed in biofilms developed in two continuous flow reactors. While biofilms harbor regions of active anabolism, the techniques also demonstrate that these biofilms contain regions of complete inactivity. Such inactive zones may contribute to the special ecology of biofilms and tolerance to antimicrobial agents. The techniques, particularly BrdU labeling, are generic and may find application to many microbial biofilm systems.
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McAdory, Louis E. "CHEMICAL SYNTHESIS, BACTERIAL EXPRESSION, AND CHARACTERIZATION OF PRO-GNRH/GAP, A PRECURSOR PROTEIN OF TWO BIOLOGICAL ACTIVE PEPTIDE HORMONES." VCU Scholars Compass, 1998. https://scholarscompass.vcu.edu/etd/5233.
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Two biologically active peptides, gonadotropin releasing hormone (GnRH) and GnRH associated peptide (GAP) are both derived from a common prohormone precursor protein, pro-GnRH/GAP. Both peptides are cosecreted from hypothalamic neurosecretory granules and are involved in the regulation of mammalian reproduction. A calcium dependent, neutral pH serine protease discovered in this laboratory, GAP-releasing enzyme, is the most likely processing enzyme of pro-GnRH/GAP. GAP-releasing enzyme is immunologically related to PC1/3, a member of the prohormone convertase (PC) class of processing endoproteinases.
GAP-releasing enzyme recognizes the eight residue processing site within pro-GnRH/GAP, G6LRPGGKR13, and correctly cleaves the R13-D14 bond to yield bioactive GAP and a three residue extension of GnRH. We and others have postulated that the recognition site for GAP-releasing enzyme forms a defined structural element at the surface of the substrate protein and that this structural element helps mediate limited endoproteolysis.
In the work reported here, hundred mg quantities of pro-GnRH/GAP were prepared by novel methods of both chemical synthesis and bacterial expression. Large amounts of pure protein are required for enzymatic and biophysical studies of pro-GnRH/GAP, which are intended to establish whether or not the processing site within the prohormone exists as a defined structural element that plays a central role in endoproteolytic processing. Synthetic pro-GnRH/GAP was prepared in high yield but proved difficult to purify to homogeneity. Recombinant pro-GnRH/GAP was prepared in sufficient yield and purity to perform all subsequent experiments.
An immunoassay was developed against a processing site epitope within pro-GnRH/GAP. Both synthetic and recombinant pro-GnRH/GAP proteins are immunoreactive, consistent with the idea that the epitope, and, thus, the processing site, is located on the surface of the molecule. Proteolysis of synthetic or recombinant pro-GhRH/GAP by trypsin or kallikrein caused immediate loss of immunoactivity, showing that the processing site is susceptible to proteolysis and that the integrity of the processing site is required for immunoactivity. One of the kallikreih hydrolytic products was identified as GAP. Therefore, kallikrein cleaves at the R13-D14 bond.
The intrinsic fluorescence yield of the Trp residue near the processing site region of pro-GhRH/GAP is sensitive to changes in pH, but not to changes in ionic strength or calcium concentration; its fluorescence yield is maximal at neutral pH. This suggests that the processing site displays maximum structure at neutral pH. This finding is coincident with the fact that GAP-releasing enzyme is optimally active at neutral pH. However, the relative contribution of secondary structural elements, as discerned by circular dichroism, remains constant over the range of pH 5.2-10.7. Only at pH
Thermal denaturation of pro-GnRH/GAP follows a simple two-state transition at neutral pH, as assessed by differential scanning calorimetry. This shows that pro-GnRH/GAP assumes a protein—like tertiary structure at neutral pH. 1D NMR data obtained at variable pH showed changes in resonance position and spectral resolution which are consistent with pH mediated conformational change and with the assumption of organized structure at neutral pH. However, the lack of through space correlations in the 2D NOESY experiment indicates that determination of the three-dimensional structure of pro-GnRH/GAP at neutral pH may be problematic.
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Eltschkner, Sandra [Verfasser], and Caroline [Gutachter] Kisker. "Targeting the Bacterial Fatty-Acid Synthesis Pathway: Towards the Development of Slow-Onset Inhibitors and the Characterisation of Protein-Protein Interactions / Sandra Eltschkner ; Gutachter: Caroline Kisker." Würzburg : Universität Würzburg, 2020. http://d-nb.info/1223851052/34.
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Sanford, Brianne. "Role of Coupled Dynamics and a Strictly Conserved Lysine Residue in the Function of Bacterial Prolyl-tRNA Synthetase and Substrate Binding by a Related trans-Editing Enzyme ProXp-ala." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397645941.
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Mas, Moruno Carlos. "Design and synthesis of peptides that neutralize bacterial endotoxins as therapeutic agents for the treatment of sepsis." Doctoral thesis, Universitat de Barcelona, 2009. http://hdl.handle.net/10803/670812.
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La sepsis es un síndrome muy complejo de definir, diagnosticar y tratar. La patología de la sepsis se deriva de una respuesta perjudicial del organismo a una infección. La exposición a endotoxinas bacterianas induce una respuesta sistémica inflamatoria que tiene como finalidad luchar contra la infección. Esta respuesta se puede describir con una serie de síntomas clínicos como la fiebre o un aumento en la frecuencia cardiaca y respiratoria, entre otros. Este proceso, necesario para la defensa del organismo, conduce a la destrucción de los patógenos. Pero si todo y activar estos mecanismos, la infección persiste así como la exposición del organismo a productos bacterianos nocivos, los síntomas clínicos descritos pueden agravarse derivando en una septicemia. Las septicemias en un gran número de casos progresan a estadios más graves, como las septicemias severas o el shock séptico, que acostumbran a comportar un fallo generalizado de órganos vitales y la muerte del paciente. Las septicemias son la primera causa de mortalidad en las unidades de cuidados intensivos, con una mortalidad que varia entre el 30 y el 70%. En los Estados Unidos, el número de pacientes con septicemia supera ya los 750.000 casos anuales, siendo la décima causa de mortalidad. Estas cifras son parecidas en Europa.
El lipopolisacárido (LPS) es la molécula que origina la septicemia y todas sus variantes, y es responsable de un gran nombre de enfermedades infecciosas. Esta molécula es el componente mayoritario de la pared celular de les bacterias Gram-negativas. La estructura química del LPS se basa en dos dominios unidos covalentemente y claramente diferenciados. Una parte hidrofílica formada por unidades repetitivas de oligosacáridos, y una parte lipídica, conocida como el lípido A que confiere toxicidad a la molécula.
La presente Tesis Doctoral explora el diseño, la síntesis y la evaluación biológica de péptidos con capacidad para neutralizar el LPS, a partir de proteínas de unión al LPS. También se examina el diseño de inhibidores de tipo peptídico, con actividad biológica y propiedades farmacocinéticas mejoradas. Finalmente se estudia una familia de peptidomiméticos, peptoides, para neutralizar el LPS. Estos compuestos resultaron ser incapaces para neutralizar esta endotoxina, pero demostraron ser citotóxicos frente diversas líneas tumorales.
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Bayart, Caroline. "Site-specific glycoconjugate synthesis." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1267.
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Les vaccins conjugués furent développés suite à l’inefficacité des vaccins polysaccharidiques chez les nourrissons et les personnes âgées. Les vaccins conjugués sont composés d’un polysaccharide extrait de la capsule bactérienne et d’une protéine porteuse. Celle-ci permet de décupler la réponse immunitaire, permettant aux vaccins d’être efficaces. L’évolution des connaissances en chimie et en analytique permettent aujourd’hui de mieux caractériser ces vaccins et de mieux maîtriser leur production. Cependant, les chimies de conjugaison utilisées pour lier le polysaccharide et la protéine porteuse, ne sont pas toujours définies et cela mène souvent à l’obtention de produits hétérogènes. Les objectifs de cette thèse ont été d’étudier le polysaccharide, les protéines porteuses et de nouvelles voies de conjugaisons pour lier spécifiquement ces deux biomolécules.Différents outils analytiques ont été utilisés afin d’acquérir une meilleure connaissance des deux partenaires de conjugaison. Cela a également permis d’établir une stratégie d’analyse efficace pour caractériser les produits de réaction. La spécificité des réactions de conjugaison a été induite par l’utilisation d’espaceurs bi-fonctionnels, réagissant spécifiquement sur certains acides aminés. Leur réactivité a d’abord été testée sur un modèle peptidique. Cela a permis de faciliter la caractérisation et d’étudier l’efficacité et la spécificité des réactions. Les réactions efficaces ont ensuite été testées différents modèles : de la protéine au vaccin. Sur les quatre réactions testées, une a été efficace sur tous les modèles. Cette chimie de conjugaison est prometteuse pour le développement de nouveaux vaccinsConjugate vaccines were developed because polysaccharide vaccines were not efficient in infant and old people. These vaccines were composed of the polysaccharide extracted from the bacterial capsule linked to a carrier protein. This protein created an immunological boost which allowed the vaccine to induce a proper protection for everyone. As chemistry knowledge and analytical techniques evolved, vaccines can now be better characterized and the production can be better controlled. Nevertheless, the chemistries used to bind the polysaccharide and the carrier protein are not always well-defined, which leads to the production of heterogeneous products. The objectives of this PhD were to study the polysaccharide, carrier proteins and new conjugation chemistries to specifically bind the two biomolecules. The other challenge was to be able to check the reaction specificity and characterize reaction products.To do so different analytical tools were used to allow a better knowledge of both conjugation partners but also to establish an efficient analytical strategy for glycoconjugate characterization. Conjugation reactions specificity was induced by using different bi-functional linkers, reacting specifically for one type of amino acid. Linkers’ reactivity was first tested on a model peptide. This allowed to facilitate the characterization and to check for both reaction specificity and reaction success. Efficient reactions were then tested on different models from carrier proteins to glycoconjugate vaccines. One of the four tested reactions was efficient from the peptide to the vaccine model. This conjugation is thus promising for the development of new conjugate vaccines
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Mendoza, J. Alexander Hoang. "Development of a codon-optimized Latrodectus hesperus MaSp1 synthetic gene for bacterial protein expression using a seamless cloning strategy." Scholarly Commons, 2015. https://scholarlycommons.pacific.edu/uop_etds/174.
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Spider silk has outstanding mechanical properties, displaying high tensile strength and extensibility. The unique combination of strength and great extensibility make it one of the toughest materials in the world. Of the seven different spider silks, dragline silk, the lifeline silk of the spider, represents one of the most renowned fiber types that has extraordinary properties. As a result, many labs across the globe are racing to manufacture synthetic dragline silk fibers. With the production of synthetic dragline silk fibers, there are unlimited commercial applications. In this study, we developed several codon-optimized MaSp1 minifibroin constructs for recombinant protein expression in bacteria. These recombinant MaSp1 minifibroin constructs were engineered to contain the N-terminal domain (NTD), different copies of internal block repeats (ranging from 2 to 64 copies of 35 amino acid blocks), and the C-terminal domain (CTD). The NTD and CTDs were derived from the natural cDNA sequences of black widow spiders, while the internal block repeats were generated from synthetic DNA fragments that were codon-optimized for expression in Escherichia coli . Different numbers of internal block repeats were created using a specialized seamless cloning strategy. By applying this seamless cloning strategy, we successfully multimerized MaSp1 block repeats that approach the natural fibroin size. Moreover, through the construction of a customized NTD-CTD spidroin construct, multimerized block repeats from any fibroin can be rapidly inserted to facilitate minifibroin protein expression in bacteria. Overall, this strategy as well as the created vectors, should help advance the silk community in the production of synthetic silk fibers that have properties that more closely resemble natural fibers.
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Kunzmann, Martin Herbert [Verfasser], Stephan A. [Akademischer Betreuer] Sieber, and Sabine [Akademischer Betreuer] Schneider. "Synthesis of gamma-lactones for activity based protein profiling: Investigation of their protein reactivity and inhibition of bacterial virulence / Martin Herbert Kunzmann. Gutachter: Sabine Schneider ; Stephan A. Sieber. Betreuer: Stephan A. Sieber." München : Universitätsbibliothek der TU München, 2014. http://d-nb.info/1047185512/34.
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Verghese, Jenson. "Investigations of Novel Mechanisms of Action for Anti-Bacterial and Anti-Cancer Agent Development." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/611.
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The development of drugs and therapeutic agents for combating infections and human malignancies continues to be a forefront area in both academic and industrial research. This is driven by the rapid emergence of multi-drug resistant bacterial strains and accumulating mutations in cancer targets that is quickly rendering our current arsenal of drugs ineffective for these therapies. Unless new drugs with novel mechanisms of action are identified and developed at a faster pace, we face a losing battle in managing these diseases. The first part of this work concerns with the natural product Simocyclinone D8 (SD8). Simocyclinone D8 is an angucyclinone antibiotic that inhibits DNA gyrase with a novel mechanism of action that has been termed competitive inhibition. Simocyclinone D8 was found to inhibit the growth of both Gram-(+ve) and Gram-(–ve) organisms and also inhibit a fluoroquinolone resistant mutant of DNA gyrase. Inspired by the structure and novel mechanism of action that SD8 displays, we synthesized analogues based on the co-crystal structure of SD8 with DNA gyrase. These compounds were found to inhibit DNA gyrase, albeit by a different mechanism of action than that of SD8. We also conducted studies towards the total chemical synthesis of SD8 and made three out of the four fragments in SD8 in decent yields. The second part of this work is focused on the development of a substrate-competitive covalent inhibitor for protein kinase B (AKT). AKT is a valid target for cancer research with two compounds currently in late stage clinical trials. Developing substrate- competitive inhibitors for kinases is a novel approach in targeting them, with very few examples in the literature. This mechanism has been postulated to overcome common resistance mutations that cancer targets harbor. A major drawback in this approach is the low binding affinity for peptide substrates by kinases. We circumvented this problem of affinity by utilizing a covalent mode of binding and synthesized a potent non-peptide active-site directed irreversible compound that inhibits AKT. Further studies on this compound are underway and are expected to yield a compound that can be used as a therapeutic agent or as a probe for AKT.
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Honda, Malca Sumire [Verfasser], and Bernhard [Akademischer Betreuer] Hauer. "Substrate characterization and protein engineering of bacterial cytochrome P450 monooxygenases for the bio-based synthesis of omega-hydroxylated aliphatic compounds / Sumire Honda Malca. Betreuer: Bernhard Hauer." Stuttgart : Universitätsbibliothek der Universität Stuttgart, 2013. http://d-nb.info/1035640430/34.
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Kao, Daniel Joseph. "Development of a synthetic peptide vaccine and antibody therapeutic for the prevention and treatment of Pseudomonas Aeruginosa infection /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2007.
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Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007.Typescript. Includes bibliographical references (leaves 203-212; 260-261). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;
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Millar, Kristina K. "Antibiotic Efficacy and Interaction in Escherichia coli during Varying Nutrient Conditions." Scholarship @ Claremont, 2016. http://scholarship.claremont.edu/scripps_theses/809.
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Due to the recent rise in antibiotic resistant pathogens, and the difficulties surrounding the quest for new antibiotics, many researchers have started revisiting antibiotic interactions in hopes of finding new treatment options. The primary outcome of this project was to examine the efficacy of concomitant antibiotic use under varying nutrient conditions, to identify variations in antibiotic interactions. Antibiotic interactions were studied, utilizing E. coli as a model bacterial system, grown in four different media types. E. coli cultures were treated with streptomycin, tobramycin, erythromycin, and amikacin individually and in a pairwise fashion at varying doses. We found that at least some antibiotic efficacies were dependent on the environmental nutrient conditions E. coli was grown in, as the antibiotics were not equally effective in all media types. E. coli grown in potato dextrose broth, in particular, showed extremely high tolerance to antibiotic inhibition. In addition, we observed several variations in antibiotic interactions, depending on the combination of antibiotics and environmental conditions utilized. It is predicted that differences in available nutrients is the primary cause of the observed discrepancies in antibiotic properties between media. The observation of changes in antibiotic efficacy under different environmental and nutrient conditions has serious implications for use of antibiotic combinations as drug treatments. Not all microenvironments within the human body have identical nutrient make-up. If the interactions antibiotics are reported to have in one environmental condition change under another, reckless prescription of combinations could lead to a serious adverse reaction. Thus, this is an important area for future in vitro and in vivo research.
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Kirchner, Alexander. "Synthese von Edelmetallclustern auf S-Layern und deren katalytische Eigenschaften." Doctoral thesis, [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=976482282.
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Fleurie, Aurore. "Rôle de la sérine-thréonine kinase StkP dans la division et la morphogenèse du pneumocoque." Thesis, Lyon 1, 2013. http://www.theses.fr/2013LYO10152.
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La bactérie Streptococcus pneumoniae peut provoquer de sérieuses pathologies chez l'homme telles que des pneumonies, méningites ou septicémies. L'étude de cette bactérie constitue donc un enjeu de santé publique international. Ces dernières années, il a été mis en évidence que les bactéries exprimaient des Sérine/Thréonine Protéine‐Kinases de type eucaryote (STPKs) et que ces dernières intervenaient dans la régulation de nombreux processus cellulaires. Une approche prometteuse serait donc de cibler les mécanismes de régulation contrôlés par les STPKs pour lutter contre les infections à pneumocoque. L'analyse du génome de S. pneumoniae a montré que cette bactérie possède un seul gène codant pour une STPK, la protéine StkP. Mes travaux de thèse ont montré que StkP est un acteur majeur de la division cellulaire et de la morphogenèse du pneumocoque. J'ai montré que son activité kinase est dépendante de la protéine GpsB et qu'elle phosphoryle spécifiquement plusieurs protéines dont la protéine de division DivIVA. L'ensemble de mes travaux permet de proposer un modèle dans lequel la triade StkP/GpsB/DivIVA régulerait finement la division et l'élongation cellulaire du pneumocoque. À plus long terme, ces travaux pourront servir de base à des études plus structurales pour développer des molécules bloquant les processus dépendants de la phosphorylation assurée par StkP, et générer ainsi de nouvelles molécules affectant le pouvoir pathogène du pneumocoqueThe bacterium Streptococcus pneumoniae is the causative agent of several diseases such as pneumonia, meningitis or septicemia. The study of this bacterium represents thus an international health challenge. Over the last decade, bacteria have been shown to produce eukaryotic‐like Serine/Threonine Protein‐Kinases (STPKs) that are involved in the regulation of several cellular processes. A promising approach would be to target the regulatory mechanisms controlled by STPKs to combat pneumococcal infections. The pneumococcus possesses a single gene encoding for a STPK, the protein StkP. The aim of my work was to characterize the biological function of StkP. My work shows that StkP plays crucial roles in the cell division and morphogenesis of S. pneumoniae. I show that the cell division protein GpsB is required for the kinase activity of StkP that, in turn, specifically phosphorylates the cell division protein DivIVA. Altogether, I propose a model in which the StkP/GpsB/DivIVA triad finely tunes S. pneumonia cell division and elongation. These data could provide the basis for future structural studies to develop specific inhibitors of StkP‐mediated phosphorylation and affecting pneumococcal virulence
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Zucchini, Laure. "Rôle du domaine extracellulaire de la sérine/thréonine-kinase StkP dans la division cellulaire et la morphogenèse du pneumocoque." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1103.
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Streptococcus pneumoniae (ou pneumocoque) est un agent pathogène humain responsable de maladies invasives et potentiellement mortelles. Les mécanismes impliqués dans le processus d'invasion restent largement inconnus, mais plusieurs observations suggèrent que les processus de signalisation impliquant la phosphorylation des protéines participeraient au caractère invasif du pneumocoque. Le génome de S. pneumoniae code pour une seule tyrosine-kinase (CpsD) et une seule sérine/thréonine-kinase (StkP). Cette dernière serait notamment impliquée dans la virulence, la compétence et la division cellulaire. Elle représente donc une cible thérapeutique potentielle intéressante pour lutter contre les infections liées au pneumocoque. L'objectif de cette thèse a donc été de caractériser le rôle de cette sérine/thréonine-kinase StkP dans la division cellulaire du pneumocoque. StkP est une protéine transmembranaire qui se caractérise par la présence de motifs structuraux conservés dans son domaine catalytique appelés motifs de Hanks. De plus, StkP possède un domaine extracellulaire composé de la répétition de quatre domaines PASTA (Penicillin-binding protein And Serine/Threonine kinase Associated). Le modèle actuel suggère que ces domaines PASTA seraient capables de fixer des fragments de la paroi cellulaire afin de permettre l'activation de StkP qui se comporterait donc comme un récepteur membranaire permettant de réguler la division cellulaire du pneumocoque. Mes travaux de thèse ont permis de revisiter ce modèle en démontrant que les domaines PASTA ne servent pas uniquement à contrôler l'activité protéine-kinase de StkP mais également à contrôler l'épaisseur de la paroi cellulaire et ainsi permettre la constriction de la cellule. Plus précisément, j'ai démontré que le domaine PASTA distal est spécialisé dans l'interaction avec une hydrolase de la paroi cellulaire alors que les trois autres domaines PASTA sont nécessaires à l'activité kinase de StkP mais également au positionnement du domaine PASTA distal. Ainsi, le domaine extracellulaire de StkP se comporterait comme une règle permettant de définir l'épaisseur de la paroi cellulaire de la bactérie. Ces travaux permettent donc de proposer un nouveau modèle d'activation et de régulation de la division cellulaire par la sérine/thréonine-kinase StkPStreptococcus pneumoniae (the pneumococcus) is one of the most important human pathogens that causes potentially fatal invasive diseases. Mechanisms required for the pneumococcal invasion process remain largely unknown, but several observations suggest that phosphorylation-based signaling processes will be at play in the invasiveness of the pneumococcus. S. pneumoniae encodes only one tyrosine-kinase (CpsD) and one serine/threonine-kinase (StkP). The latter would be involved in virulence, competence, and cell division. StkP represent therefore a promising target to combat pneumococcal infections. My aims were to better understand the role of StkP in pneumococcal cell division. StkP is a transmembrane protein characterized by the presence of a series of conserved structural motifs called Hanks motifs in its catalytic domain. In addition, StkP possesses an extracellular domain composed of the repetition of four PASTA domains (Penicillin-binding protein And Serine/Threonine kinase Associated). The current model proposes that PASTA domains are able to bind cell wall fragments resulting in StkP kinase activation. StkP would thus behave as an authentic kinase receptor regulating cell division. My thesis works has allowed to revisit this model by showing that PASTA domains do not only serve StkP kinase activation. Rather, they contribute to determine the cell wall thickness and govern cell constriction. More precisely, I demonstrated that the distal PASTA domain possesses unique features for the binding of a cell wall hydrolase whereas the other three contributes to StkP kinase activation and the positioning of the distal PASTA domain. Thus, the extracellular domain of StkP acts as a ruler determining the cell wall thickness. This work allows to propose an alternative model of activation and regulation of cell division by the serine/threonine-kinase StkP
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Wychowski, Czeslaw. "Expression de la proteine de capside vp1 du poliovirus dans les bacteries et dans les cellules animales : identification d'un epitope de neutralisation et caracterisation de sequences indispensables a l'accumulation de proteines dans le noyau." Paris 7, 1987. http://www.theses.fr/1987PA077173.
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Manuse, Sylvie. "Régulation de la morphogenèse et de la division cellulaire du pneumocoque par phosphorylation : rôle de la sérine / thréonine kinase StkP et des protéines DivIVA, GpsB et MapZ." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10342.
Full textAbstract:
Malgré les modèles établis pour certaines bactéries, la morphogenèse de bactéries de formes atypiques est peu comprise. C'est le cas de la bactérie pathogène pour l'homme Streptococcus pneumoniae, ou pneumocoque, qui possède une forme ovo-diplococcale. Cependant, à mon arrivé au laboratoire, il avait été démontré qu'une sérine/thréonine protéine-kinase membranaire appelée StkP était indispensable à la division cellulaire et à la morphogenèse du pneumocoque. L'objectif de ma thèse a ainsi été de caractériser certains substrats de StkP et d'étudier leur rôle, ainsi que l'impact de leur phosphorylation, au cours du processus de division cellulaire. Dans ce contexte, j'ai montré que le substrat DivIVA et son paralogue GpsB coordonnent l'élongation et la division cellulaire du pneumocoque. Ces travaux permettent de proposer un nouveau modèle de morphogenèse du pneumocoque dans lequel la triade StkP/DivIVA/GpsB organise la synthèse de la paroi cellulaire nécessaire à l'élongation et à la division de la cellule. J'ai également mis en évidence que la protéine MapZ interagit avec la paroi cellulaire lors de l'élongation cellulaire afin de marquer de manière permanente le site de division, où elle recrute la protéine FtsZ. Ces travaux ont ainsi permis d'identifier un système inédit de régulation positive du positionnement du site de division chez les bactéries. Enfin, j'ai caractérisé les déterminants moléculaires du positionnement de MapZ au centre de la cellule. S. pneumoniae étant un pathogène humain important, nous pouvons anticiper que nos données pourraient servir de base fondamentale à des projets plus appliqués de lutte contre les infections bactériennesDespite the established models for some bacteria, the morphogenesis of bacteria with atypical shapes is poorly understood. This is the case of the human pathogen Streptococcus pneumoniae, or pneumococcus, that displays an ovo-diplococcal shape. However, when I joined the lab, it had just been shown that a membrane serine/threonine kinase named StkP was crucial for the cell division and the morphogenesis of the pneumococcus. The goal of my thesis was to characterize the substrates of StkP and to study their function as well as the impact of their phosphorylation in the cell division process. First, I have shown that the substrate DivIVA together with its paralog GpsB coordinate cell elongation and division of the pneumococcus. Based on these observations, we propose a new model of pneumococcal morphogenesis in which the triad StkP/DivIVA/GpsB organizes cell wall synthesis involved in cell elongation and division. In a second part of my work, I have studied another substrate of StkP that was of unknown function and that we named MapZ. I have shown that MapZ interacts with the cell wall during the cell elongation to position at midcell. Then MapZ recruits the cell division protein FtsZ and controls the closure of the Z-ring. This work has uncovered a new mechanism of positive regulation for the positioning of the division site in bacteria. Finally, I characterized the molecular determinants of MapZ positioning at the division site. S. pneumoniae is an important human pathogen, we can thus anticipate that our work will represent a fundamental base for applied projects in order to develop new strategies against bacterial infections
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Kaminishi, Tatsuya, Andreas Schedlbauer, Attilio Fabbretti, Letizia Brandi, Lizarralde Borja Ochoa, Cheng-Guang He, Pohl Milon, Sean R. Connell, Claudio O. Gualerzi, and Paola Fucini. "Crystallographic characterization of the ribosomal binding site and molecular mechanism of action of Hygromycin A." Oxford University Press, 2015. http://hdl.handle.net/10757/608247.
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Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC.Bizkaia:Talent and the European Union's Seventh Framework Program (Marie Curie Actions; COFUND; to S.C., A.S., T.K.); Marie Curie Actions Career Integration Grant (PCIG14-GA-2013-632072 to P.F.); Ministerio de Economía Y Competitividad (CTQ2014-55907-R to P.F., S.C.); FIRB Futuro in Ricerca from the Italian Ministero dell'Istruzione, dell'Universitá e della Ricerca (RBFR130VS5_001 to A.F.); Peruvian Programa Nacional de Innovación para la Competitividad y Productividad (382-PNICP-PIBA-2014 (to P.M. and A.F.)). Funding for open access charge: Institutional funding.
Revisión por pares
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Diez, Simon. "Guanosine nucleotides link cell wall metabolism and protein synthesis during entry into quiescence." Thesis, 2021. https://doi.org/10.7916/d8-ppqb-da53.
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Quiescence, a transitory period of non-growth, is a ubiquitous aspect that is present in all organisms. In addition to being present in all forms of life, quiescence is a feature that has been observed in cells that are important for human health, including stem cells in mammals and antibiotic tolerant cells in bacteria. In bacteria, quiescence per se has recently been suggested to underlie the transient tolerance to a wide range of antibiotics. Furthermore, most microbial life exists in a quiescent state. Despite their prevalence and importance, relatively little is known about the physiology of quiescent bacteria. One aspect of bacterial quiescence that has been repeatedly observed is their lowered metabolic activity compared to actively growing bacteria. How do cells that grow and divide enter into a temporary state of non-growth? In particular, how are the energy-intensive processes that are required for growing cells regulated during a non-growing state? The main subject of this thesis is to investigate how protein synthesis, the most energy-intensive process in growing bacterial cells, is regulated during entry into a quiescent phenotype (stationary phase).
I first investigate how protein synthesis is regulated using a single cell method that fluorescently tags nascent polypeptide chains. In chapter 3, I show that during entry into stationary phase, protein synthesis is downregulated heterogeneously with one group of cells having comparatively low protein synthesis, resulting in a population that is approximately bimodal. I further show that this bimodality is dependent on a signaling system (PrkC and its partner phosphatase PrpC) that senses cell wall metabolism. I connect signaling from this system to the expression of an enzyme (SasA) that produces a group of nucleotides that are major regulators of growth in bacteria ((pp)pGpp). Lastly, I show that the bimodality is dependent on the three enzymes that synthesize (pp)pGpp.
In chapter 4, I explore in detail how the bimodality in protein synthesis is generated. This heterogeneity requires the production of (pp)pGpp by three synthases: SasA, SasB, RelA. I first show that these enzymes differentially affect this bimodality: RelA and SasB are necessary to generate the sub-population exhibiting low protein synthesis, whereas SasA is necessary to generate cells exhibiting comparatively higher protein synthesis. The RelA product (pppGpp) allosterically activates SasB, and I find that the SasA product (pGpp) competitively inhibits this activation. I provide in vivo evidence that this antagonistic interaction mediates the observed heterogeneity in protein synthesis. This chapter, therefore, identifies the mechanism underlying the generation of phenotypic heterogeneity in the central physiological process of protein synthesis.
In chapter 5, I next turn to understand the biochemical mechanism by which cells with comparatively low levels of protein synthesis down-regulate this process. I first show that ppGpp is sufficient to inhibit protein synthesis in vivo. I then show that ppGpp inhibits protein synthesis by inhibiting translation initiation directly by binding to the essential GTPase, Initiation Factor 2 (IF2). In collaboration with Ruben Gonzalez’s lab, we also show that ppGpp prevents the allosteric activation of IF2. Finally, I demonstrate that the observed attenuation of protein synthesis during the entry into quiescence is a consequence of the direct interaction of (pp)pGpp and IF2.
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Huang, Bridget Yih Jiin. "Regulation of Release Factor 2 in Non-canonical Translation Pathways." Thesis, 2017. https://doi.org/10.7916/D80P10MC.
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Protein synthesis, or translation, is a complex, multi-step process that requires regulatory and quality control mechanisms to ensure the accurate production of proteins. Two major challenges during bacterial protein synthesis are maintaining the accuracy of translation during the elongation stage and resolving stalled ribosomal complexes. Interestingly, bacteria have evolved two mechanisms, a post-peptidyl transfer quality control (post PT QC) and a ribosome rescue mechanism, to counter these challenges. Both of these mechanisms make use of a protein factor that normally functions during translation termination, Release Factor 2 (RF2), along with an additional protein factor, Release Factor 3 (RF3) for post PT QC and Alternative ribosome-rescue factor A (ArfA) for ribosome rescue, to achieve these non-canonical functions. The mechanistic role of RF3 and ArfA in these two pathways remains unclear; however, they may play a role in regulating RF2 in context of these non-canonical pathways. As a step toward understanding the role of RF3 and ArfA in post PT QC and ribosome rescue and, in particular, their role in the regulation of RF2, I sought to determine the effect of RF3 and ArfA on the binding kinetics of RF2 in post PT QC and ribosome rescue pathways. Using a single-molecule fluorescence resonance energy transfer (smFRET) signal between the P-site peptidyl-tRNA and RF2, the binding and dissociation of RF2 can be directly monitored in the absence or in the presence of RF3 or ArfA.
In Chapter 2, I describe the development of smFRET signals using different chromophores, cyanine 3 to cyanine 5 (Cy3-Cy5) or to a fluorescence quencher (Cy3-QSY9). The Cy3-Cy5 and Cy3-QSY9 smFRET signals complement each other for monitoring RF2 binding; whereas Cy3-Cy5 is suitable for observing stable binding using low substrate concentration, Cy3-QSY9 is suitable for observing transient binding using high substrate concentration. The RF2 binding and dissociation to ribosomal complexes was first examined in the absence of other factors thus providing the foundation for studying the regulation of RF2 binding by RF3 or ArfA.
In the bacterial post PT QC mechanism, RF3 enhances the rate of RF2-mediated peptide release to catalyze premature termination of miscoded protein, thus ultimately increasing the fidelity of protein synthesis1. Without addition of RF3, the rate of RF2-mediated peptide release is too slow to compete with the rate of protein synthesis. In Chapter 3, the role of RF3 on RF2 binding kinetics in post PT QC was investigated using both an fMet-Lys-tRNALys(Cy3) to RF2(Cy5) smFRET signal and an fMet-Lys-tRNALys(Cy3) to RF2(QSY9) smFRET signal.
The ArfA-RF2 ribosome rescue pathway is a backup mechanism for trans-translation, which relieves stalled ribosomal complexes by providing an open reading frame coding for both a degradation tag and a stop codon2. Because the expression of ArfA is under strict control by trans-translation, the ArfA-RF2 pathway only functions in the absence of active trans-translation. More importantly, deletion of both the trans-translation and ArfA-RF2 pathways leads to synthetic lethality in E. coli, highlighting the critical role of ribosome rescue in vivo3. In Chapter 4, I used an fMet-Phe-tRNAPhe(Cy3) to RF2(Cy5) smFRET signal to evaluate the role of ArfA on RF2 binding and dissociation in the ribosome rescue pathway. Collectively, these studies survey the regulation of RF2 binding kinetics by RF3 or ArfA in performing non-canonical functions such as post PT QC and ribosome rescue in bacteria.