Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

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Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the {delta}-Al-{var_epsilon} activation domain from c-jun and the Drosophila RNA-binding protein Sex-lethal. The second system is the Drosophila Sex-lethal (Sxl) protein, an RNA-binding protein which is the ``master switch`` in sex determination. Sxl contains two adjacent RNA-binding domains (RBDs) of the RNP consensus-type. The NMR spectrum of the second RBD (Sxl-RBD2) was assigned using multidimensional heteronuclear NMR, and an intermediate-resolution family of structures was calculated from primarily NOE distance restraints. The overall fold was determined to be similar to other RBDs: a {beta}{alpha}{beta}-{beta}{alpha}{beta} pattern ... continued below

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266 p.

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Lee, A.L. May 1, 1996.

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  • Lee, A.L. Lawrence Berkeley National Lab., CA (United States). Structural Biology Div.

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Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the {delta}-Al-{var_epsilon} activation domain from c-jun and the Drosophila RNA-binding protein Sex-lethal. The second system is the Drosophila Sex-lethal (Sxl) protein, an RNA-binding protein which is the ``master switch`` in sex determination. Sxl contains two adjacent RNA-binding domains (RBDs) of the RNP consensus-type. The NMR spectrum of the second RBD (Sxl-RBD2) was assigned using multidimensional heteronuclear NMR, and an intermediate-resolution family of structures was calculated from primarily NOE distance restraints. The overall fold was determined to be similar to other RBDs: a {beta}{alpha}{beta}-{beta}{alpha}{beta} pattern of secondary structure, with the two helices packed against a 4-stranded anti-parallel {beta}-sheet. In addition {sup 15}N T{sub 1}, T{sub 2}, and {sup 15}N/{sup 1}H NOE relaxation measurements were carried out to characterize the backbone dynamics of Sxl-RBD2 in solution. RNA corresponding to the polypyrimidine tract of transformer pre-mRNA was generated and titrated into 3 different Sxl-RBD protein constructs. Combining Sxl-RBD1+2 (bht RBDs) with this RNA formed a specific, high affinity protein/RNA complex that is amenable to further NMR characterization. The backbone {sup 1}H, {sup 13}C, and {sup 15}N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and {sup 15}N relaxation experiments were carried out to characterize the backbone dynamics of this complex. The changes in chemical shift in Sxl-RBD1+2 upon binding RNA are observed using Sxl-RBD2 as a substitute for unbound Sxl-RBD1+2. This allowed the binding interface to be qualitatively mapped for the second domain.

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266 p.

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OSTI as DE96014961

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  • Other Information: TH: Thesis (Ph.D.)

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  • Other: DE96014961
  • Report No.: LBNL--39117
  • Grant Number: AC03-76SF00098
  • Office of Scientific & Technical Information Report Number: 373861
  • Archival Resource Key: ark:/67531/metadc675707

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Office of Scientific & Technical Information Technical Reports

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  • May 1, 1996

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  • July 25, 2015, 2:20 a.m.

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  • April 5, 2016, 10:50 a.m.

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Lee, A.L. Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution, thesis or dissertation, May 1, 1996; California. (digital.library.unt.edu/ark:/67531/metadc675707/: accessed October 15, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.