A conserved histidine in switch-II of EF-G moderates release of inorganic phosphate

Abstract

Elongation factor G (EF-G), a translational GTPase responsible for tRNA-mRNA translocation possesses a conserved histidine (H91 in Escherichia coli) at the apex of switch-II, which has been implicated in GTPase activation and GTP hydrolysis. While H91A, H91R and H91E mutants showed different degrees of defect in ribosome associated GTP hydrolysis, H91Q behaved like the WT. However, all these mutants, including H91Q, are much more defective in inorganic phosphate (Pi) release, thereby suggesting that H91 facilitates Pi release. In crystal structures of the ribosome bound EF-G•GTP a tight coupling between H91 and the γ-phosphate of GTP can be seen. Following GTP hydrolysis, H91 flips ~140° in the opposite direction, probably with Pi still coupled to it. This, we suggest, promotes Pi to detach from GDP and reach the inter-domain space of EF-G, which constitutes an exit path for the Pi. Molecular dynamics simulations are consistent with this hypothesis and demonstrate a vital role of an Mg2+ ion in the process.

Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome

“…In translational GTPases, switch loops I and II are involved in the GTPase activity (reviewed in Voorhees and Ramakrishnan, (2013) ). Switch loop II (aa 105–110), which carries the catalytic H108 (H92 in E. coli EF-G; ( Cunha et al, 2013 ; Holtkamp et al, 2014 ; Koripella et al, 2015 ; Salsi et al, 2014 ) is well resolved in all five structures. The histidine resides next to the backbone of G3028 of the sarcin-ricin loop and near the diphosphate of GDP ( Figure 5e ).…”

EF-G Activation by Phosphate Analogs

“…Several independent reports suggested that substituting GTP with non-hydrolysable analogues of GTP only moderately (by 2–3 fold) slow down the rate of mRNA translocation[17, 18]. Amino acid substitutions in the G domain of EF-G that inhibited the GTPase activity of EF-G reduced the rate of mRNA/tRNA translocation by only 7 to 30 fold[12, 19–21]. Furthermore, a number of antibiotics, such as viomycin or hygromycin B, strongly inhibit translocation by binding to the ribosome without impeding the binding of EF-G, GTP hydrolysis or P i release[1, 22, 23].…”

Selective translation by alternative bacterial ribosomes

“…The crude ribosome suspension was further digested with MNase at 4°C overnight. After secondary digestion, the ribosome preparation was dialyzed against HEPES-polymix buffer (59, 60). The ribosome preparation was incubated with anti-Flag M2 beads 4°C overnight.…”

“…Initiation complex was formed by incubating 70S ribosome variants as described above (0.2 μM), [ 3 H]fMet-tRNA fMet (0.5 μM), XR7mRNA (AUG-UAA) (0.5 μM), initiation factors (0.5 μM) and GTP (1 mM) at 37°C for 15 min in HEPES-polymix buffer (59). The reactions were thereafter filtered through BA-85 nitrocellulose membranes and washed with 10ml of ice-cold HEPES-polymix buffer (pH 7.5).…”