Steady-state and stopped-flow fluorescence measurements of the interaction of translational initiation factors withmRNA and the interaction of transcription stimulatory factor USF with DNA.
Item
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Title
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Steady-state and stopped-flow fluorescence measurements of the interaction of translational initiation factors withmRNA and the interaction of transcription stimulatory factor USF with DNA.
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Identifier
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AAI9605660
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identifier
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9605660
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Creator
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Sha, Ma.
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Contributor
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Adviser: Dixie J. Goss
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Date
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1995
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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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The binding of oligoribonucleotides or m{dollar}\sp7{dollar}GTP to wheat germ protein synthesis initiation factor eIF-4B, eIF-(iso)4F and the purified subunits (p28, 28 kDa and p86, 86 kDa) of eIF-(iso)4F were measured by direct fluorescence techniques. Analysis of the equilibrium association constants (Keq) indicates that eIF-4B RNA binding is not affected by the m{dollar}\sp7{dollar}GTP cap structure or the AUG initiation codon of translation. eIF-4B is insensitive to hairpin structures within the oligoribonucleotide. The binding site size of mRNA on eIF-4B is approximately 18 bases. A specific anion effect of Cl{dollar}\sp-{dollar} on eIF-4B binding to oligoribonucleotides was found when comparing the ionic strength effect of KC{dollar}\rm\sb2H\sb3O\sb2{dollar} and KCl. The binding of m{dollar}\sp7{dollar}GTP to p28 as a function of pH, temperature and ionic strength is described. Iodide quenching shows that all 9 tryptophan residues in p28 are exposed while only 6 out of 16 tryptophan residues are exposed on the intact eIF-(iso)4F. As in the eIF-(iso)4F protein, a specific anion effect of Cl{dollar}\sp-{dollar} on p28 binding to m{dollar}\sp7{dollar}GTP was found. The binding of p28, p86, and eIF-(iso)4F with m{dollar}\sp7{dollar}GTP and mRNA analogues was measured and compared. The second part of this thesis was devoted to the study of transcription upstream stimulatory factor USF. USF is a human transcriptional activation factor, which uses a basic/helix-loop-helix/leucine zipper (b/HLH/Z) motif to form a homotetramer and recognize specific sequences in the promoter region of both nuclear and viral genes transcribed by RNA polymerase II. Steady-state fluorescence spectroscopy demonstrated that the b/HLH/Z domain of USF binds to its DNA targets with high affinity and specificity, whereas removal of the leucine zipper yielding the b/HLH minimal DNA-binding region reduces both affinity and specificity. Stopped-flow measurements provided kinetic evidence for a two-step binding process, involving rapid formation of a protein-DNA intermediate followed by a slow isomerization step. Titration studies revealed that the first binding event has an equilibrium constant K{dollar}\sb{lcub}\rm eq{rcub}{dollar} = (2.2 {dollar}\pm{dollar} 2.0) {dollar}\times{dollar} 10{dollar}\sp9{dollar}M{dollar}\sp{lcub}-1{rcub}{dollar} for MLP (Adenovirus Major Late Promoter) DNA, whereas the second binding occurs with a remarkably reduced affinity K{dollar}\sb{lcub}\rm eq{rcub}{dollar} = (1.2 {dollar}\pm{dollar} 0.8) {dollar}\times{dollar} 10{dollar}\sp8{dollar}M{dollar}\sp{lcub}-1{rcub}.{dollar} This anti-cooperative feature of DNA binding by the homotetramer suggests that USF stimulates transcription by mediating DNA looping between nearby recognition sites located in nuclear and viral gene promoters.
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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.
