1. Both nucleotide-binding sites must close for efficient 70S splitting.
Both nucleotide-binding sites must close for efficient 70S splitting. (A) A minimal set of splitting factors required for active splitting comprises ABCE1, aRF1, and aIF6 from S. solfataricus. All factors were pure and monodisperse as shown by SDS–PAGE (Coomassie), immunoblotting, and SEC. (B) Specific 70S splitting requires aRF1, ABCE1, and AMP-PNP. Results were normalized to the highest splitting ratio for WT ABCE1 with AMP-PNP and aRF1. (C) Traces corresponding to (B) clearly demonstrate an increase of 50S in the presence of WT ABCE1, aRF1, and AMP-PNP. (D) Ribosome splitting is most efficient, when both sites are in a closed, occluded state. Colors as in (B). (E) No ribosomes are actively split by any of the SR “disengagement” mutants, showing that closure of both sites is a prerequisite for ribosome splitting. Colors as in (B). Representative set of three independent experiments.
Elina Nürenberg-Goloub et al. LSA 2018;1:e
© 2018 Nürenberg-Goloub et al.