Lost in translation: An inside look at translation regulation of tobacco etch virus RNA.
Item
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Title
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Lost in translation: An inside look at translation regulation of tobacco etch virus RNA.
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Identifier
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AAI3330129
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identifier
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3330129
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Creator
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Yumak, Hasan.
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Contributor
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Adviser: Dixie J. Goss
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Date
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2008
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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 | Biology, Virology
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Abstract
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Zeenko and Gallie (2005, J.B.C; 280, 26813-26824) showed that the pseudoknot (PK1) of tobacco etch virus (TEV) RNA is necessary to promote cap-independent translation. We showed the first quantitative data (24) on the binding interactions of plant protein synthesis initiation factors with IRES and demonstrated that the TEV leader can discriminate between eIF4G isoforms to preferentially recruit one isoform over the other. We studied the effect of Poly(A)-binding protein (PABP) on the PK1 RNA binding to eIF4F in the presence and absence of eIF4B. Equilibrium studies of PK1 RNA binding to eIF4F showed ∼ 2-fold stronger affinity in the presence of eIF4B. Addition of eIF4B and PABP to the eIF4F enhances binding affinity ∼ 4-fold as compared to eIF4F binding to PK1 RNA. Further, we investigated the effect of Poly(A)20 on the binding of initiation factors to PK1 RNA. In the presence of Poly(A)20, eIF4F˙PABP bound to PK1 about 4-fold tighter. Further, addition of Poly (A)20 enhances the binding affinity of eIF4F˙eIF4B˙PABP protein complex by about 3-fold for PK1 RNA. Overall, eIF4F˙eIF4B˙PABP˙Poly(A) 20 complex has 11-fold higher affinity to PK1 RNA as compared with binding affinity of eIF4F alone. Kinetic analysis of eIF4F and eIF4F˙4B with PK1 RNA were also measured and compared. The stopped-flow fluorescence anisotropy measurements demonstrated that the observed rate constant for the binding of PK1 RNA increased linearly with an increase in eIF4F and eIF4F˙4B initiation factor concentration. The association rate constant (kon) for PK1 binding was ∼ 2-fold faster for eIF4F˙4B than for eIF4F alone. eIF4F-PK1 RNA complex shows a slower rate of dissociation in the presence of eIF4B (k off = 10.2 +/- 0.7 s-1 for eIF4F˙4B; k off = 11.6 +/- 0.3 s-1 for eIF4F). Since viral protein synthesis is simpler than host cell protein synthesis, the information obtained from this research has potential use to develop systems to produce desired proteins which are nutritionally beneficial and have other economic uses.
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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.
