Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

Abstract

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

Review: Translational GTPases

“…In particular, the interaction of the phosphate group of A2662 with the side chain of His84 is likely responsible for the stabilization of the rotated conformation of His84 towards the nucleotide in the activated state . This is in agreement with the results of atomic mutagenesis studies, where replacement of one of the two nonbridging phosphate oxygens of A2662 with a methyl group resulted in a complete loss of GTPase stimulation of EF‐G by the ribosome . Also Asp21 might take part in the stabilization of the TS by SRL, which would explain why this residue is present in trGTPases, but not in Ras‐like GTPases.…”

“…20 This is in agreement with the results of atomic mutagenesis studies, where replacement of one of the two nonbridging phosphate oxygens of A2662 with a methyl group resulted in a complete loss of GTPase stimulation of EF-G by the ribosome. 95 Also Asp21 might take part in the stabilization of the TS by SRL, 58 which would explain why this residue is present in trGTPases, but not in Ras-like GTPases. Recent high-resolution structures of the ribosome-bound EF-G show the side chain of the respective Asp residue coordinating a Mg 21 ion close to the crucial A2662, indicating that the GTPase-activated state might also involve a rearrangement of Asp21.…”

Structure of the GTP Form of Elongation Factor 4 (EF4) Bound to the Ribosome

“…Structural studies have revealed that nucleotide A2662 of the SRL places the catalytic His-84 residue in EF-Tu and His-87 in EF-G into their activated positions (16,17,29). Recently, a more detailed role for A2662 was revealed by the atomic mutagenesis approach; namely, that the non-bridging phosphate oxygen of A2662 participates in the activation of GTP hydrolysis on trGTPases (30). Here, A2662 in the SRL is shown to interact with the catalytic His-86 residue to place it into its activated position, further corroborating a universal mechanism of trGTPase activation through the SRL.…”

Ribosomal protein L7/L12 is required for GTPase translation factors EF‐G, RF3, and IF2 to bind in their GTP state to 70S ribosomes

“…Ribosome‐dependent GTPases bind to the prokaryotic 70S ribosome complex through both the sarcin‐ricin loop (SRL) and the GTPase‐associated center (GAC) . Recent structural and mechanistic data support a model of GTPase activation, whereby a phosphate oxygen from A2662 in the SRL is a strong determinant for GTP hydrolysis . By contrast, the role of the GAC is less understood with several ambiguous findings.…”