Eukaryotic initiation factor 4F (elF4F) enhances high affinity binding of 40S ribosomal subunit to tobacco etch virus (TEV) internal ribosome entry site (IRES)
Item
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Title
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Eukaryotic initiation factor 4F (elF4F) enhances high affinity binding of 40S ribosomal subunit to tobacco etch virus (TEV) internal ribosome entry site (IRES)
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Identifier
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d_2009_2013:fb4c9b29dd86:10703
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identifier
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10783
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Creator
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Yumak, Sumeyra,
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Contributor
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Dixie J. Goss
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Date
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2010
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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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Biochemistry | Biophysics | FLUORESCENCE | INITIATION FACTORS | RIBOSOME | RNA | TEV
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Abstract
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Many viruses and some eukaryotic mRNAs employ a cap-independent pathway in which an RNA element called the internal ribosome entry site (IRES), drives preinitiation complex formation by positioning the ribosome on the message, either at or just upstream of the start site. We have studied binding of wheat germ 40S subunit of wheat germ ribosome to wild type PK1 RNA, which shows 100 % translation activity. To explore the specificity of the IRES RNA•40S interaction, we also measured the affinity for IRES RNA mutants, S2-3 RNA and to S1-3 RNA that have been previously characterized in terms of in vitro translation initiation activity (1). The level of expression from each mutant is calculated relative to the corresponding wild-type sequence, which is set at 100%.S2-3 RNA shows about 30% translation activity while S1-3 RNA shows about 7% translation activity.;While ribosome binding to eukaryotic mRNAs requires the participation of eukaryotic initiation factors eIF4A, eIF4B, and eIF4F and the hydrolysis of ATP, our results suggest that the 40S ribosomal subunit binds directly to the PK1 RNA with dissociation constant (Kd) of 67 nM. While 40S ribosome bound to S2-3 RNA with Kd of 138 nM; significant binding to S1-3 RNA was not observed. Direct binding measurements of both wild type and mutant TEV IRES RNAs suggest that TEV message is driven through a high affinity TEV RNA•40S interaction, involving a novel strategy of ribosome recruitment distinct from eukaryotic cap-dependent mechanisms.;Fluorescence anisotropy data showed that eIF4F increased 40S Ribosome binding affinity to PK1 RNA by approximately 2.6 fold. To determine the effects of initiation factor mediated RNA unwinding in cap independent initiation, we determined RNA binding activity of elF4A, elF4B and 40S Ribosome with TEV RNAs. The fluorescence anisotropy data revealed that in the presence of eIF4A, eIF4B, and hydrolysis of ATP, binding affinity of 40S ribosome to PK1 RNA increased 4 fold; giving Kd of 17 nM. Under the same conditions, while binding affinity of 40S ribosome to S2-3 RNA increased 2 fold, there was no significant change in S1-3 RNA binding to 40S.These data correlate well with the observed translational data and provide more detailed information on the translational strategy of tobacco etch virus.
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Type
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dissertation
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Source
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2009_2013.csv
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degree
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Ph.D.
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Program
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Chemistry
