conserved GTPase HflX is a ribosome splitting factor that binds to the E-site of the bacterial ribosome

Nucleic Acids Research, Feb 2016

Coatham, Mackenzie L., Brandon, Harland E., Fischer, Jeffrey J., Schümmer, Tobias, Wieden, Hans-Joachim

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conserved GTPase HflX is a ribosome splitting factor that binds to the E-site of the bacterial ribosome

The conserved GTPase HflX is a ribosome splitting factor that binds to the E-site of the bacterial ribosome Mackenzie L. Coatham 0 Harland E. Brandon 0 Jeffrey J. Fischer 0 Tobias Schu¨ mmer 0 Hans-Joachim Wieden 0 0 Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge , Lethbridge, Alberta, T1K 3M4 , Canada 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. INTRODUCTION Translation is an essential ribosome-mediated process in all cell types that occurs in four sequential phases: initiation, elongation, termination and recycling. For efficient polypeptide synthesis, additional ribosome associated proteins are required at each of these phases. 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. Each of the previously mentioned GTPases have been characterized to be involved in at least one of the phases of translation, yet some, like EF-G, function during both elongation and recycling. Furthermore, 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 ( 2–4 ), stress response ( 5,6 ) and release of tetracycline from the bacterial ribosome ( 7– 10 ), respectively). Structures of these translational GTPases (trGTPases) share common structural features mimicking, to various degrees, the structure of tRNA ( 11–13 ). For example, the structure of EF-G•GDP shares a common shape with the ternary complex of EF-Tu•GTP•aminoacyl-tRNA ( 14,15 ). Furthermore, cryo-electron microscopy (cryo-EM) reconstructions of ribosome-bound EF-Tu•GTP•aa-tRNA (16), EF-G ( 17,18 ), LepA ( 3,4 ), BipA ( 19 ) and Tet(O)/TetM ( 10,20 ), indicate a common binding site for the translational GTPases in the ribosomal A-site. The recent crystal structure of EF-Tu•GTP•aa-tRNA bound to the 70S ribosome revealed that this GTPase activation likely occurs through the correct positioning of the catalytic histidine residue at the end of switch II (DxxGH) by A2662 of the sarcin-ricin loop (SRL), allowing a nucleophilic attack by a water molecule on the -phosphate of bound GTP (21). This mechanism has been proposed to be shared amongst trGTPases ( 21 ). The universally conserved protein HflX ( 22–24 ), whose GTPase activity is also enhanced significantly by 50S and 70S ribosomal particles ( 22 ), provides an exception to the above common features of trGTPases. The X-ray crystal structure of HflX from Sulfolobus solfataricus reveals that the N-terminus of the factor is unique, with no identifiable structural homolog ( 25 ). The Escherichia coli homolog of HflX is a three-domain protein consisting of the unique N-terminal HflX-domain, a central G-domain and a Cterminal domain not found in the archaeal S. solfataricus homolog (Figure 1A). Additionally, E. coli HflX has a 22 amino acid N-terminal extension (Figure 1B). These extensions at the termini of HflX are not unique to the E. coli protein, but are found in most bacteria and eukaryotes at varying lengths. Several studies have studied truncations of these domains and found reduced binding to the ribosome and differences in nucleotide preference ( 26 ). Furthermore, knockout strains of HflX are viable, yet are more susceptible to high intercellular levels of manganese ( 27 ). In E. coli the gene encoding HflX is found downstream of the gene for Hfq, the universal stress response protein in bacteria, and both are under the control of a heat sensitive promoter ( 28,29 ). In an effort to elucidate the bind (...truncated)


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Coatham, Mackenzie L., Brandon, Harland E., Fischer, Jeffrey J., Schümmer, Tobias, Wieden, Hans-Joachim. conserved GTPase HflX is a ribosome splitting factor that binds to the E-site of the bacterial ribosome, Nucleic Acids Research, 2016, pp. 1952-1961, Volume 44, Issue 4, DOI: 10.1093/nar/gkv1524