Research Strategy – Approach
Specific Aim 1: How does the primary sequence of MEX-5 determine its RNA-binding specificity?
The CCCH-type TZF proteins recognize their RNA targets though a template of H-bonds between the amide and carbonyl groups of the protein backbone and the Watson-Crick edges of the bases (ref Hudson, Teplova). This observation suggests that in CCCH-type TZF proteins any variation of the primary sequence that results in a change of the backbone conformation alters the pattern of interactions between the protein and the RNA molecules and therefore the ability to bind specific sequences of RNA.
TIS11d is a CCCH-type TZF protein that shares 40% of sequence identity with ...view middle of the document...
1.1 Is the spacing between CCCH residues or the length of the linker between the ZFs affecting the RNA-binding specificity of MEX-5?
Strategy: The CCCH-type TZF domain of MEX-5 (residues 268-346) will be expressed and purified to perform RNA-binding studies. Residues Gly282 (in ZF1) and Thr328-Gly329 (in ZF2) will be deleted in turn from the MEX-5 sequence to generate the variants MEX-5ZF1, MEX-5ZF2 and MEX-5ZF1,ZF2 in which the original spacing between the CCCH residues is altered in order to resemble the scheme CX8CX5CX3H of TIS11d. The role of the length of the linker between the two ZFs will be investigated by producing the MEX-5 variants in which residues Asp305, Ala306, Pro307, Asn312 and Asn313 are deleted in turn. The candidates for deletion have been chosen with particular attention to maintain the charged and aromatic moieties that are conserved between MEX-5 and TIS11d (specifically, Arg309 and Tyr310 in the linker).
The variants of MEX-5 will be characterized using electrophoretic mobility shift assays (EMSA) and fluorescence anisotropy (FP) to measure the affinity to the optimal target sequence of TIS11d (UUUUAUUUAUUUU, ARE13) labeled with fluorescein at the 3' . To determine variations in RNA-binding specificity among the variants, a competition assay will be used to measure the affinities to mutated sequences of ARE13. The mutations of ARE13 will consist in substitution of the two adenosine to pyrimidine and in variations of the spacing of the two adenosine in the original sequence.
Results from EMSA and FP with labeled ARE13 will be analyzed by fitting the data with a modified form of the Hill equation to compensate deviations from ideal conditions (ref. Ryder2010):
where m and b are normalization factors, n is the Hill's coefficients and [Pt] is the total concentration of protein in the sample. For competition reactions, a quadratic solution of the Lin and Riggs equation (ref) determined by Weeks and Crothers will be used (ref.), where in turn fitted parameters will be held constant or allowed to float during the fit as it is appropriate. The fraction of the labeled RNA in presence of an unlabeled competitor is accordingly estimated as:
where m and b are normalization factors, Kd and Kc are dissociation constants for labeled RNA and unlabeled competitor, C, P and R are respectively the concentration of competitor, protein and labeled RNA.
Engineer variants of MEX-5268-346 using Quickchange site-directed mutagenesis.
Expression and purification of soluble MEX-5268-346 and variants following a protocol adapted from Wright2004.
Expression and purification of MEX-5268-346 and variants from inclusion bodies using a protocol adapted from Wright2004.
Determine binding affinities of MEX-5268-346 and variants to fluorescein-labeled ARE13 as described in Pagano.
Determine binding affinities of MEX-5268-346 and variants to unlabeled ARE13 mutants using competition EMSA and FP assays.
Expected outcomes and...