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Gene Synthesis> | N-terminal His-tagged RuvBL1 and untagged RuvBL2 were cloned as indicated in Lopez-Perrote et al.28. For pull-down experiments, a 3xMyc tag was incorporated to the N-terminus of RUVBL1. The RPAP3 full length (FL) was purchased from GenScript. | Get A Quote |
The R2TP/Prefoldin-like co-chaperone, in concert with HSP90, facilitates assembly and cellular stability of RNA polymerase II, and complexes of PI3-kinase-like kinases such as mTOR. However, the mechanism by which this occurs is poorly understood. Here we use cryo-EM and biochemical studies on the human R2TP core (RUVBL1-RUVBL2-RPAP3-PIH1D1) which reveal the distinctive role of RPAP3, distinguishing metazoan R2TP from the smaller yeast equivalent. RPAP3 spans both faces of a single RUVBL ring, providing an extended scaffold that recruits clients and provides a flexible tether for HSP90. A 3.6 Å cryo-EM structure reveals direct interaction of a C-terminal domain of RPAP3 and the ATPase domain of RUVBL2, neces... More