Characterizing The M Rna Binding Activity Of Mex 5

1747 words - 7 pages

Specific Aim 2: What is the structure of the TZF domain of MEX-5 and what is its contribution to the RNA-binding specificity?
The known structures of the CCCH-type TZF proteins highlight the fact that despite the presence of highly conserved residues in the primary sequence, these domains assume diverse conformations. In TIS11d the linker region is in an extended conformation, and the two ZFs are oriented in a parallel way. In MBNL-1, instead, the linker participates in β-sheet formation and rigidly maintains an anti-parallel orientation of the two ZFs. The significance of the alignment of the subdomains arises from its contribution to determine the structures of the cognate RNAs: the target ...view middle of the document...

Then, it will be possible to compute the number of intermolecular and intramolecular H-bonds, van der Waals contacts and salt bridges, as well as evaluate steric constraints.
Methodology:
Expression and purification of MEX-5268-346 as in Aim 1
Determination of the crystallization condition for MEX-5268-346/ARE13 complex using the Microlytic MCSG screening suite (384 crystallization conditions).
X-Ray diffraction data collection.
Determination of the three-dimensional structure by model building and refinement.
Expected outcomes and results: Because of differences in the primary sequence between MEX-5 and other CCCH-type TZF proteins (mainly the spacing between the CCCH residues and the length of the linker), it is expected that the unique specificity of MEX-5 to RNA sequences arise from a novel structure of the ZFs. The three-dimensional structure of the TZF domain of MEX-5 will allow for the determination of the protein backbone architecture that allow patterns of interactions (H-bonds and intermolecular stacking) with RNA molecules that differ with respect to TIS11d and MBNL-1.
Potentials problems and alternative strategies: One potential issue concerns the possibility that could not be possible to find the crystallization condition for the protein-RNA complex. This can be due to instability of the complex, degradation of the RNA along the period required or limitation to the protein concentration in the sample due to aggregation. If this is the case, the three-dimensional structure of the protein-RNA complex will be calculated using molecular dynamics (MD) simulations and NOE (nuclear Overhauser effect) and torsion angle restraints from NMR spectroscopy experiments as described in Hudson2004. Although previous works (ref) have shown that local structures of each ZF can be well defined using this method, the determination of the relative orientation of the two non-interacting ZFs could be challenging because of the predicted elongated shape of the complex. To address this problem, RDC (residual dipolar coupling) restraints will be included in the calculations.
Specific Aim 3: How does the electrostatic interaction between MEX-5 and RNA contribute to RNA-binding specificity?
Nucleic acids are highly negatively charged molecules because of the phosphodiester backbone, thus in many protein-RNA complexes a dominant role in association is played by electrostatic interactions. Although sequence-specific proteins contact single stranded RNA (ssRNA) via the bases, previous studies (ref. Law2006) demonstrated that interactions between charged residues and the nucleic acid backbone contribute significantly to RNA binding even at distances up to 11 Å. The electrostatic contribution, however, does not provide sequence-specificity: the charge distribution of the ssRNA, in fact, is independent of the sequence.
For MEX-5, the total calculated charge for the TZF domain at neutral pH is +9.4 e.u., whereas for TIS11d and MBNL-1 (ZF3/4), which are more specific...

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