Translation Elongation Factor 4 (LepA) Contributes to Tetracycline Susceptibility by Stalling Elongating Ribosomes

Abstract

Even though elongation factor 4 (EF4) is the third most conserved protein in bacteria, its physiological functions remain largely unknown and its proposed molecular mechanisms are conflicting among previous studies. In the present study, we show that the growth of an Escherichia coli strain is more susceptible to tetracycline than its EF4 knockout strain. Consistent with previous studies, our results suggested that EF4 affects ribosome biogenesis when tetracycline is present. Through ribosome profiling analysis, we discovered that EF4 causes 1-nucleotide shifting of ribosomal footprints on mRNA when cells have been exposed to tetracycline. In addition, when tetracycline is present, EF4 inhibits the elongation of protein synthesis, which leads to the accumulation of ribosomes in the early segment of mRNA. Altogether, when cells are exposed to tetracycline, EF4 alters both ribosome biogenesis and the elongation phase of protein synthesis.

Omnipresent Maxwell's demons orchestrate information management in living cells

“…In fact, it modulates the initiation cycle of protein synthesis. It is also involved in ribosome 30S subunit biosynthesis (Liu and Chen, ). The informational role of EF4 is witnessed by the fact that mRNA molecules possessing strong ribosome‐binding sites tend to be translated with reduced efficiency in a ΔlepA background (Balakrishnan et al ., ).…”

A conserved translation factor is required for optimal synthesis of a membrane protein family in mycobacteria

“…To examine the link between a ribosomal factor and the phenotypes we observed, we first hypothesized that, like other LepA homologs [33, 41, 48], mycobacterial LepA functions as a ribosome-associated GTPase. To test whether the GTPase activity of LepA was critical to the phenotypes observed in lepA -cells, we assayed calcein staining and drug killing with a GTPase-null lepA allele ( lepA H109A).…”

“…To define LepA-related features of the porin, within either the mRNA sequence or the protein sequence, we took advantage of the fact that LepA affects the abundance of MspA-C and not MspD. Previous LepA ribosome profiling data from E.coli implicated the presence of certain glycine codons in transcripts with an altered translation rate [41, 42]. We found a 2-3 fold increase in the use of the glycine codon ‘GGT’ in mspA-C relative to mspD (Supplementary Figure 3A).…”

“…The C-terminal domain of LepA makes contact with the A/P-site tRNA and, likely, alters positioning of the tRNA species on the ribosome [33–35]. While it is non-essential in most bacteria, in some species, LepA might confer some fitness benefit in certain growth conditions, such as altered cation concentrations or low pH [36–41]. Despite its conservation, the physiological role of LepA remains unclear.…”

“…Two different functions have been proposed for LepA. In E. coli , loss of LepA results in decreased polysome formation as a consequence of altered initiation rate and ribosome assembly defects [34, 41, 42]. Alternatively, structural studies of multiple bacterial LepA homologs indicate that while it binds the same site as EF-G on the 70S ribosome, it may alter the conformation of the ribosome-tRNA complex [43–45] and, perhaps, participate in translational quality control.…”