Kinetics of the interactions between yeast elongation factors 1A and 1B alpha, guanine nucleotides, and aminoacyl-tRNA

The interactions of elongation factor 1A (eEF1A) from Saccharomyces cerevisiae with elongation factor 1B alpha (eEF1B alpha), guanine nucleotides, and aminoacyl-tRNA were studied kinetically by fluorescence stopped-flow. eEF1A has similar affinities for GDP and GTP, 0.4 and 1.1 mu M, respectively. Dissociation of nucleotides from eEF1A in the absence of the guanine nucleotide exchange factor is slow ( about 0.1s(-1)) and is accelerated by eEF1B alpha by 320-fold and 250-fold for GDP and GTP, respectively. The rate constant of eEF1B alpha binding to eEF1A (10(7)-10(8) M-1 s(-1)) is independent of guanine nucleotides. At the concentrations of nucleotides and factors prevailing in the cell, the overall exchange rate is expected to be in the range of 6 s(-1), which is compatible with the rate of protein synthesis in the cell. eEF1A center dot GTP binds Phe-tRNA(Phe) with a K-d of 3 nM, whereas eEF1A center dot GDP shows no significant binding, indicating that eEF1A has similar tRNA binding properties as its prokaryotic homolog, EF-Tu.