Abstract
The universally conserved P-loop GTPases control diverse cellular processes, like signal transduction, ribosome assembly, cell motility, and intracellular transport and translation. YchF belongs to the Obg-family of P-loop GTPases and is one of the least characterized member of this family. It is unique because it preferentially hydrolyses ATP rather than GTP, but its physiological role is largely unknown. Studies in different organisms including humans suggest a possible role of YchF in regulating the cellular adaptation to stress conditions. In the current study, we explored the role of YchF in the model organism Escherichia coli. By western blot and promoter fusion experiments, we demonstrate that YchF levels decrease during stress conditions or when cells enter stationary phase. The decline in YchF levels trigger increased stress resistance and cells lacking YchF are resistant to multiple stress conditions, like oxidative stress, replication stress, or translational stress. By in vivo site directed cross-linking we demonstrate that YchF interacts with the translation initiation factor 3 (IF3) and with multiple ribosomal proteins at the surface of the small ribosomal subunit. The absence of YchF enhances the anti-association activity of IF3, stimulates the translation of leaderless mRNAs, and increases the resistance against the endoribonuclease MazF, which generates leaderless mRNAs during stress conditions. In summary, our data identify YchF as a stress-responsive regulator of leaderless mRNA translation.
Highlights
Throughout their life, cells need to flexibly respond to changes in their environment
YchF and its eukaryotic homolog Ola1 belong to the Obg group and are the least characterized GTPases (Balasingam et al, 2020), there is accumulating evidence for their involvement in ribosome-associated processes (Becker et al, 2012; Samanfar et al, 2014; Chen et al, 2015; Ding et al, 2016) and stress response (Zhang et al, 2009; Cheung et al, 2010; Wenk et al, 2012; Chen et al, 2015; Hannemann et al, 2016)
A common response to stress conditions in eukaryotes and prokaryotes is the downregulation of ribosomal proteins and ribosomal RNA (rRNA) (Dennis and Nomura, 1974; Warner, 1999; Lemke et al, 2011; Starosta et al, 2014), which reduces protein synthesis and prevents the formation of misfolded or damaged proteins (Deuerling and Bukau, 2004; Tyedmers et al, 2010; Cherkasov et al, 2013; Brandman and Hegde, 2016); we did not find any indication that E. coli YchF had a significant influence on synthesis or assembly of ribosomes under stress conditions and YchF does not appear to act as a regulator of ribosome production
Summary
Throughout their life, cells need to flexibly respond to changes in their environment. Sophisticated sensing and signal transduction systems have evolved in both eukaryotic and prokaryotic cells These systems allow adjusting cell physiology in response to environmental and internal cues, and promote cell survival under stress conditions (Starosta et al, 2014). Stress responses primarily involve changes in gene expression and result in metabolic alterations, modification of enzymatic activities, and changes in protein homeostasis (de Nadal et al, 2011). The latter includes the adjustable synthesis of stressresponse proteins, of which many are universally conserved and considered to constitute the minimal stress proteome. The TRAFAC class of proteins comprises a functionally heterogeneous group of proteins, which include translation factors (Rodnina and Wintermeyer, 2016) and protein targeting factors (Steinberg et al, 2018), as well as proteins involved in ribosome assembly (Sato et al, 2005), cell cycle regulation (Foti et al, 2007), and stress response (Kuo et al, 2008)
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