Synthesis of a fluorescentmRNA cap analogue and a study of cap structure recognition, guanosine nucleotide binding and exchange, and helicase activity in wheat germ protein synthesis initiation.
Item
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Title
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Synthesis of a fluorescentmRNA cap analogue and a study of cap structure recognition, guanosine nucleotide binding and exchange, and helicase activity in wheat germ protein synthesis initiation.
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Identifier
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AAI9707146
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identifier
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9707146
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Creator
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Ren, Jianhua.
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Contributor
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Adviser: Dixie J. Goss
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Date
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1996
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Analytical | Chemistry, Biochemistry | Biology, Molecular
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Abstract
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For the study of the cap binding reaction, one approach is to use fluorescence spectroscopy. A ribose modified fluorescent cap analogue, Ant-m{dollar}\sp7{dollar}GTP, was designed and synthesized for this purpose. This fluorescent cap analogue was found to have a high quantum yield, resistance to photobleaching, and avoided overlapping of excitation and emission wavelength with those of proteins. The binding of Ant-m{dollar}\sp7{dollar}GTP with wheat germ initiation factors eIF-4F and eIF-(iso)4F was measured. The fluorescent cap analog was found to react with cap binding protein with similar affinity with m{dollar}\sp7{dollar}GpppG. The microenvironment of Ant-m{dollar}\sp7{dollar}GTP when bound to protein was tested by fluorescence quenching experiments. The second portion is devoted to study of the recycling of wheat germ eIF-2 in protein synthesis initiation. A direct fluorescence spectroscopic study on the binding of wheat germ eIF-2 with GDP and GTP was performed. Results indicated that dissociation constants for wheat germ eIF-2{dollar}\cdot{dollar}GTP and eIF-2{dollar}\cdot{dollar}GDP were more similar than for mammalian eIF-2. A substitution experiment was performed and it was found that wheat germ eIF-2 bound GDP can be readily exchanged with free GTP at a relatively low GTP/GDP concentration ratio. This suggests the possibility that in the wheat germ system, guanosine nucleotide exchange may be regulated by controlling the GTP/GDP concentration ratio and may not require an analog of the mammalian guanosine nucleotide exchange factor eIF-2B. In the third major portion, the interaction of two wheat germ eukaryotic protein synthesis initiation factors, eIF-(iso)4F and eIF-4A, with mRNA has been examined for helicase activity. It was found that eIF-4A and eIF-(iso)4F could unwind the double stranded RNA. ATP and Mg{dollar}\sp{lcub}2+{rcub}{dollar} were required for this reaction. The reaction was cap-dependent and required a single stranded region. These studies combined with binding affinity studies suggest that the cap-binding subunit of eIF-(iso)4F forms a complex with mRNA. Subsequently, eIF-4A is bound. Hydrolysis of ATP provides energy for the protein complex to unwind the double-stranded region.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
