Abstract
Using a combination of biochemical, structural probing and rapid kinetics techniques we reveal for the first time that the universally conserved translational GTPase (trGTPase) HflX binds to the E-site of the 70S ribosome and that its GTPase activity is modulated by peptidyl transferase centre (PTC) and peptide exit tunnel (PET) binding antibiotics, suggesting a previously undescribed mode of action for these antibiotics. Our rapid kinetics studies reveal that HflX functions as a ribosome splitting factor that disassembles the 70S ribosomes into its subunits in a nucleotide dependent manner. Furthermore, our probing and hydrolysis studies show that the ribosome is able to activate trGTPases bound to its E-site. This is, to our knowledge, the first case in which the hydrolytic activity of a translational GTPase is not activated by the GTPase activating centre (GAC) in the ribosomal A-site. Furthermore, we provide evidence that the bound state of the PTC is able to regulate the GTPase activity of E-site bound HflX.
Highlights
Translation is an essential ribosome-mediated process in all cell types that occurs in four sequential phases: initiation, elongation, termination and recycling
Using light scattering in conjunction with stopped-flow rapid kinetics, we show that HflX is able to split the ribosome in a nucleotide-dependent manner, similar to that of elongation factors (EFs)-G and RRF during ribosome recycling
The localization of HflX’s binding site to the tRNA exit site of the 70S ribosome makes HflX the first known trGTPase to bind at that location and whose guanosine -triphosphatases (GTPases) activity is stimulated by the ribosome
Summary
Translation is an essential ribosome-mediated process in all cell types that occurs in four sequential phases: initiation, elongation, termination and recycling. Several of the involved proteins function as guanosine 5 -triphosphatases (GTPases), utilizing the hydrolysis of GTP to drive their functional cycle. These factors include the canonical and essential translation factors initiation factor (IF) 2, elongation factors (EFs) Tu and G, and release factor (RF) 3. There are additional ribosomeassociated GTPases not essential for translation including: the EF-Tu homolog SelB, which is responsible for delivery of selenocysteinyl-tRNA to the elongating ribosome [1], the EF-G homologs LepA, BipA, and ribosome protection proteins (RPPs) such as Tet(O) and TetM (responsible for reverse translocating the ribosome [2,3,4], stress response [5,6] and release of tetracycline from the bacterial ribosome [7,8,9,10], respectively)
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