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Study of Assembly and Function of the DrrAB ComplexPradhan, Prajakta A 30 November 2008 (has links)
The DrrAB proteins of Streptomyces peucetius belong to the ABC family of ubiquitous membrane transporters. The DrrA and DrrB proteins together form a drug efflux pump that carries out the transport of the anticancer drug doxorubicin by carrying out ATP hydrolysis. The present study is the first where the intrinsic factors involved in the assembly of the DrrAB functional complex have been elucidated. The drrA and drrB genes in the wild type operon have overlapping stop and start codons (ATGA) which indicates translational coupling between the two genes. On insertion of a fortuitous stop codon in DrrA it was shown that the expression of DrrB is coupled to that of the upstream gene drrA. Furthermore, it was observed that a functional complex could be achieved only when the genes were maintained in cis in a translationally coupled manner. Translational regulation in DrrA was found to be involved in the control of optimal levels of DrrB. Inhibitory interactions within drrA sequence were speculated to cause translational arrest at the C terminus of DrrA. A novel assembly domain that forms the interface between DrrA containing the Nucleotide Binding Domain (NBD) and DrrB comprising the TransMembrane Domain (TMD) was found. Based on the data presented in this study a model is proposed for the biogenesis of the DrrAB drug pump. The model suggests that translational coupling between DrrA and DrrB is crucial for functional complex formation. Further, there is evidence of regulation of translation by attenuation in the intergenic region of drrA and drrB. The regulation seems to involve the last 30 nucleotides of the mRNA of drrA and some upstream sequences within drrA that cause translational arrest within the C terminus of DrrA. Since DrrB is translationally coupled to drrA, this translational arrest in conjunction with coupling causes lowering in the levels of DrrB. Finally, since the DrrA-DrrB interaction domain lies in the C terminus of DrrA, only the fully translated DrrA product will be competent to form a complex with DrrB. This interaction between the C terminus of DrrA and the N terminus of DrrB may be crucial for initial targeting of the complex to the membrane. The model is expected to serve as primer and open up an interesting yet insufficiently understood subject of membrane protein biogenesis.
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Investigations of the function of the Pit-accessory protein (Pap) in Sinorhizobium melilotiTiller, Lauren January 2019 (has links)
Phosphate (PO4-3 or Pi) is an essential molecule necessary for sustaining life and it plays important roles in nucleic acid and cell membrane integrity. However, phosphate is found in growth-limiting concentrations in most environments. Bacteria have developed a diverse set of transport systems to uptake and scavenge phosphate from their environment for use in cellular processes. In the soil bacterium, Sinorhizobium meliloti, one such Pi transport system is the Pap-Pit system. Pit is a membrane transporter for Pi and is associated with a cytosolic protein of unknown function known as Pap (Pit-accessory protein). Interestingly, the stop codon of pap overlaps with the start codon of pit by a single nucleotide. In previous work, the pap gene appeared to be required immediately upstream of pit in an operon for functional Pi transport. Thus, in a pap deletion mutant, when pap is present in trans, there is no Pi transport. This suggests a possible translational coupling mechanism between Pap and Pit, in which the translation of Pap is required for the translation of Pit. Here, an alkaline phosphatase (phoA/lacZ) and a β-glucuronidase (gusA) translational reporter were fused to Pit as a measure of its translation and to understand the role of translational coupling in the Pap-Pit system. Growth complementation experiments with a conditionally Pi transport deficient S. meliloti mutant carrying various mutations in both pap and pit have also been performed in an attempt to determine the function of Pap in Pi uptake. The results presented here provide evidence that pap and pit are translationally coupled, and this is necessary for functional Pi transport via Pap-Pit. / Thesis / Master of Science (MSc) / Microbes require phosphorus in the form of inorganic phosphate (Pi) as an essential nutrient, but it is often found in growth-limiting concentrations in the environment. Bacteria have developed a diverse set of Pi transport systems to scavenge and take up phosphate from the environment. In the soil bacterium, Sinorhizobium meliloti, one such Pi transport system is the Pap-Pit system. Pit is a membrane transporter for Pi and is associated with a cytosolic protein of unknown function known as Pap. Various mutations in both pap and pit have been constructed in an attempt to determine the function of Pap in Pi uptake via Pit. The pap gene appears to be required immediately upstream of pit in an operon for functional Pi transport. The pap and pit genes overlap by a single nucleotide and this may suggest a translational coupling mechanism that is required for functional Pi transport via Pap-Pit.
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Theoretical Investigation of Biological Networks Coupled via Bottlenecks in Enzymatic ProcessingOgle, Curtis Taylor 06 June 2016 (has links)
Cell biology is a branch of science with a seemingly infinite abundance of interesting phenomena which are essential to our understanding of life and which may potentially drive the development of technology that improves our lives. Among the open ended questions within the field, an understanding of how gene networks are affected by limited cellular components is both broad and rich with interest. Common to all cellular systems are enzymes which perform many tasks within cells without which organisms could not remain healthy. Here are presented several explorations of enzymatic processing as well as a tool constructed for this purpose. More specifically, these works consider the effect of coupling of gene networks via competition for enzymes found within the cell. It is shown that a limitation on the number of available enzymes permits the formation of bottlenecks which drastically affect molecular dynamics within cells. These effects potentially afford cell behaviors that in part explain the impressive robustness of life to constantly fluctuating environments. / Ph. D.
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Mechanisms of translational regulation in bacteriaBentele, Kajetan 21 August 2013 (has links)
Diese Arbeit untersucht den Zusammenhang zwischen Mechanismen der translationalen Regulation und der Genomorganisation in Bakterien. Der erste Teil der Arbeit analysiert die Beziehung zwischen der Translationseffizienz von Genen und der Häufigkeit bestimmter Codons am Genanfang. Es ist bekannt, dass die Häufigkeitsverteilung der Codons am Anfang der Gene bei einigen Organismen eine andere ist als sonst im Genom. Durch die systematische Analyse von ungefähr 400 bakteriellen Genomen, evolutionären Simulationen und experimentellen Untersuchungen sind wir zu dem Schluss gekommen, dass die beobachtete Abweichung der Codonhäufigkeiten wohl eine Konsequenz der Notwendigkeit ist, RNA Sekundärstruktur in der Nähe des Translationsstarts zu vermeiden und somit eine effiziente Initiation der Translation zu gewährleisten. Im zweiten Teil der Arbeit untersuchen wir den Einfluss der Genreihenfolge innerhalb eines Operons auf die Fitness von E. coli. In bakteriellen Genomen vereint ein Operon funktionell zusammengehörige Gene, die in einer mRNA zusammen transkribiert werden und somit in der Expression stark korreliert sind. Daneben kann die translationale Kopplung, d. h. die Interdependenz der Translationseffizienz zwischen benachbarten Genen innerhalb einer solchen mRNA, eine bestimmte Proteinstöchiometrie weiter stabilisieren. Mithilfe eines Modells für die translationale Kopplung sowie für den Chemotaxis Signalweg konnten wir zeigen, dass die native Genreihenfolge eine der Permutationen ist, die am meisten zur Robustheit der Chemotaxis beitragen. Die translationale Kopplung ist daher ein wichtiger Faktor, der die Anordnung der Gene innerhalb des Chemotaxis Operon bestimmt. Diese Arbeit zeigt, dass die Anforderungen einer effizienten Genexpression sowie die Robustheit wichtiger zellulärer Funktionen einen Einfluss auf die Organisation eines Genoms haben können: einerseits bei der Wahl der Codons am Anfang der Gene, andererseits auf die Ordnung der Gene innerhalb eines Operons. / This work investigates the relationship between mechanisms of translational regulation and genome organization in bacteria. The first part analyzes the connection between translational efficiency and codon usage at the beginning of genes. It is known for some organisms that usage of synonymous codons at the gene start deviates from the codon usage elsewhere in the genome. By analyzing about 400 bacterial genomes, evolutionary simulations and experimental investigations, we conclude that the observed deviation of codon usage at the beginning of genes is most likely a consequence of the need to suppress mRNA structure around the ribosome binding site, thereby allowing efficient initiation of translation. We investigate further driving forces for genome organization by studying the impact of gene order within an operon on the fitness of bacterial cells. Operons group functionally related genes which are transcribed together as single mRNAs in E. coli and other bacteria. Correlation of protein levels is thus to a large extent attributed to this coupling on the transcriptional level. In addition, translational coupling, i.e. the interdependence of translational efficiency between neighboring genes within such a mRNA, can stabilize a desired stoichiometry between proteins. Here, we study the role of translational coupling in robustness of E. coli chemotaxis. By employing a model of translational coupling and simulating the underlying signal transduction network we show that the native gene order ranks among the permutations contributing most to robustness of chemotaxis. We therefore conclude that translational coupling is an important determinant of the gene order within the chemotaxis operon. Both these findings show that requirements for efficient gene expression and robustness of cellular function have a pronounced impact on the genomic organization, influencing the local codon usage at the beginning of genes and the order of genes within operons.
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