Eukaryotic Initiation Factor 2 (eIF2) is a heterotrimeric (alpha, beta, gamma) p

Question

Eukaryotic Initiation Factor 2 (eIF2) is a heterotrimeric (alpha, beta, gamma) protein complex that delivers the Met-tRNA_i to the AUG start codon during translational initiation. The left panel depicts an SDS-PAGE of purified eIF2 from three different yeast strains that express Hiss tagged versions of a wild-type, a Y142H mutation, or a K250R mutation of the gamma subunit. Crude extracts were purified through a Ni^+2-.Agarose column followed by a Heparin column. The right panel depicts an equilibrium binding assay of Met-tRHA_i to the various purified eIF2 complexes, (adapted from Erickson and Hannig. EMBO. 1996-5(22):6311-6320.) Which step in the purification procedure removed contaminating proteins that may contain structural motifs that are similar to a Hiss tag? What is the estimated purity of the wild-type eIF2? What is the estimated MW of eIF2 gamma? What is the estimated MW of eIF2 gamma? Based upon the data, what specific role does the amino acids K250 and Y142 in eIF2 gamma function?|

Explanation / Answer

Based on the given data,

a)

Running of the Crude sample in the “Ni+2-agarose column” is the purification procedure that removed contaminating proteins that may contain structural motifs that are similar to His8 tag.      

b)

The estimated purity of wild type elF2 is 99.9%

c)

The estimated Molecular weight of elF2 is 50-55 KD

d)

The estimated Molecular weight of elF2 is 120-130 KD

e)

The K250 increases the binding affinity of elF2 toward its ligand Met-tRNA, whereas Y142 decreases the affinity of elF2 for its ligand.

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