A physical characterization of eucaryotic initiation factor 4E.
Item
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Title
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A physical characterization of eucaryotic initiation factor 4E.
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Identifier
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AAI9917649
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identifier
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9917649
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Creator
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Friedland, Diana Elaine.
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Contributor
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Advisers: Dixie J. Goss | Gerald Koeppl
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Date
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1999
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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, Biochemistry | Biophysics, General | Biology, Molecular
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Abstract
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A characterization of eIF-4E was achieved using several techniques. Identification of the amino acid residues involved in the eukaryotic mRNA cap binding domain of mammalian eIF-4E was achieved using the photoaffinity analogue [gamma- 32P] 8-N3GTP. Amino acid sequencing identified the binding domain as the region containing the sequence Trp 113-Arg 122. Lys 119 was not identified in sequencing analysis nor was it cleaved by trypsin. These results indicate that Lys 119 is the residue directly modified by photoinsertion of [gamma-32P]8-N3GTP. Direct fluorescence techniques were used to measure the equilibrium constant of the recombinant eIF-4E used in this study. This was done in order to confirm that the recombinant protein had biological and physical characteristics that are similar or identical to native eIF-4E. This information was used to synthesize a peptide consisting of the eIF-4E binding site domain. Fluorescence measurements were used to determine the equilibrium activity of the peptide which was two orders of magnitude greater than that of the native, intact protein. The peptide was then used for the purpose of developing an affinity peptide column for use in isolating globin mRNA. To further identify the significance of these amino acids in mRNA cap binding and to determine their functional roles, fluorescence and circular dichroism studies were performed on reIF-4E in which the individual amino acids between Arg 112 and Gln 121 were mutated to alanine. The analyses of these mutant proteins indicates that mutants of eIF-4E of varying cap affinity can be engineered.
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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.
