The conserved protein EF4 (LepA) modulates the elongation cycle of protein synthesis

Abstract

EF4 (LepA), a strongly conserved protein, is important for bacterial growth and functional protein biosynthesis under certain conditions and is quite similar structurally to the translocase EF-G. The elongation cycle in protein synthesis is characterized by ribosome oscillation between pretranslocation (PRE) and posttranslocation (POST) complexes. Here, using ensemble single turnover and equilibrium experiments, as well as single molecule FRET measurements, we demonstrate that EF4 can compete with EF-G for binding to the PRE complex. Such EF4 binding results in formation of a complex, denoted X 3 , that effectively sequesters a catalytically active ribosome, leading to a transient inhibition of elongation that provides a mechanism for optimization of functional protein synthesis. Earlier [Liu H, et al. (2010) J Mol Biol 396:1043-1052 we demonstrated that EF4 also reacts with POST complex, leading to the formation of a complex, I 3 , that appears to be identical with X 3 . Our present results strongly suggest that PRE complex is the principal target of EF4 action on translation, rather than POST complex as had been previously supposed.

Conserved GTPase LepA (Elongation Factor 4) functions in biogenesis of the 30S subunit of the 70S ribosome

“…However, we and others have been unable to confirm this activity (4)(5)(6). Using toeprinting, we measured the rate of spontaneous reverse translocation in many contexts and found no evidence that LepA can speed the reaction (4).…”

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

“…In support of this view, single-turnover experiments and single-molecule FRET measurements reveal that EF4 prefers to bind to the PRE state ribosomes and that this interaction occurs in a competitive fashion with EF-G (12), suggesting that the PRE complex is the main substrate of EF4 during translation. The interaction between EF4 and the PRE complex gives rise to a ribosome state that can be rapidly converted to the POST complex by EF-G (12). Therefore, it is possible that the EF4-mediated PRE complex-like state arises from its interaction with the PRE complex rather than back-translocation of the POST complex (12).…”

“…The interaction between EF4 and the PRE complex gives rise to a ribosome state that can be rapidly converted to the POST complex by EF-G (12). Therefore, it is possible that the EF4-mediated PRE complex-like state arises from its interaction with the PRE complex rather than back-translocation of the POST complex (12). Curiously, Fredrick and co-workers (13) reported that EF4 failed to promote back-translocation under various conditions, including those in the previously reported mRNA toeprinting assay (2).…”

West syndrome caused by homozygous variant in the evolutionary conserved gene encoding the mitochondrial elongation factor GUF1

“…32 In these suboptimal environments, Guf1/LepA activity remobilizes stuck ribosomes and transiently inhibits the elongation process to optimize protein synthesis. 36,37 We therefore assessed complementation on plates supplemented with potassium tellurite, magnesium or low levels of chloramphenicol. Under these conditions, respiratory growth of the oxa1ΔC Δguf1 double mutant was strictly dependent on the expression of the human Guf1 protein (Figure 3).…”

“…Thus tellurite exposure blocks protein synthesis in a GUF1-dependent manner in agreement with the notion that LepA counteracts protein synthesis to prevent the misincorporation of non-cognate amino acids in stressful conditions. 36,37 The improved growth observed with the p.(A609S) variant suggests that this variant is less proficient than wild-type GUF1 in this stalling and hence possibly in limiting misincorporation.…”